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PhD project opportunities

On this page you can find out about PhD opportunities currently available in Biomedical Science. These projects cover developmental biology, cell biology, physiology and pharmacology, neuroscience, models of human disease, human stem cell biology and regenerative medicine.

Click on a project title below to find out more. Some of these projects come with funding to cover your tuition fees and living expenses, but they may not be available to students from outside the UK or the European Union.

Do you have your own idea for a project, or do you have your own funding in place?

Find a potential supervisor by visiting our research themes and centres webpages. Especially if you have your own funding in place we strongly encourage you to look at our science, and find a research area that excites you.

Contact a member of academic staff to find out about PhD opportunities in their area.

If you are applying for a project that does not come with funding, or you are not eligible for a funded project, there may be other ways for you to fund your PhD. We have a limited number of departmental scholarships available each year, and we also accept applications from students who are able to fund themselves.

It is a good idea to contact the supervisor of any PhD opportunity you want to apply for, before you submit your application. If the project you want to apply for does not come with funding, they may also be able to advise you on other sources of funding.

Once you have identified a project, a supervisor and a source of funding, you can complete the University's postgraduate online application form.


Projects for entry starting academic year 2018-19

Application deadline: 1 December 2017. Interviews to commence January 2018.

An integrated molecular, cell and proteomic analysis of palmitoylation in neurons  - Mark Collins (COMPETITION FUNDED)

Supervisor: Dr Mark Collins

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Palmitoylation, the only known reversible lipid modification of proteins, is an important regulator of protein localisation and function. It affects a plethora of cellular processes including protein trafficking, stability and signalling and is therefore important for all cell types, and organisms from yeast to humans. The extent to which this reversible post-translational modification is employed and how the specificity of the palmitoylation machinery is encoded remains to be determined.

Palmitoylation is mediated by a family of 23 enzymes (protein acyl transferases (PATs)) in humans. Many of these enzymes have been shown to regulate important aspects of cell biology and in particular in neuronal cells where palmitoylation of receptors and associated proteins regulates synaptic plasticity and therefore functions such as learning and memory. Indeed, several of these enzymes have been implicated in the pathophysiology of neurological disorders from Huntington’s disease to intellectual disability and schizophrenia. It is not well understood which proteins are modified by individual PATs in cells, how they can selectively recognise substrate proteins and what effect palmitoylation has on the function of these proteins.

This project will use proteomic approaches that we have recently developed to identify substrates of protein acyl transferases and investigate how palmitoylation of selected substrates regulates their stability, trafficking and function. This interdisciplinary project will utilise a range of experimental approaches including cell culture, molecular and cell biology, biochemistry and state-of-the-art quantitative mass spectrometry.

The student will be given in-depth training in all of these methods and will benefit from collaborations with other groups within the department. This research will further our understanding of how proteins dynamically associate with membranes and how this affects their function in health and disease.

References

  • Mark O. Collins Keith Woodley and Jyoti S. Choudhary. Global, site-specific analysis of neuronal protein S-acylation. Scientific Reports. 2017. Jul 5;7(1):4683.
  • Fukata Y, Fukata M. (2010). Protein palmitoylation in neuronal development and synaptic plasticity. Nature Reviews Neuroscience. Mar;11(3):161-75.
  • Jones ML, Collins MO, Goulding D, Choudhary JS & Rayner JC (2012). Analysis of protein palmitoylation reveals a pervasive role in Plasmodium development and pathogenesis. Cell Host & Microbe, 12(2), 246-258.
  • Cox J, Mann M. (2011). Quantitative, high-resolution proteomics for data-driven systems biology. Annual Review of Biochemistry 2011;80:273-99.

Keywords: Biochemistry, Cell Biology / Development, Genetics, Molecular Biology, Neuroscience/Neurology

Contact information

For informal enquiries about the project or application process, please feel free to contact:

Regulation of synaptic protein function by lysine acetylation - Mark Collins (COMPETITION FUNDED)

Supervisor 1: Dr Mark Collins

Supervisor 2: Dr Vincent Cunliffe

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Recent proteomic studies have identified several thousand proteins in biochemically purified synapses and have uncovered multi-protein complexes essential for synapse function. Many of these synaptic genes are associated with >100 neurological diseases, highlighting the need for a better a molecular understanding of synapses.

Synaptic activity is regulated by a number of post-translational modifications such as protein phosphorylation. Recent studies have shown that acetylation regulates the localisation of synaptic scaffolding proteins and the surface expression of a major neurotransmitter receptor. The majority of proteins present in mouse/zebrafish synapses are acetylated but almost nothing is known about its function or the mechanism of regulation.

This project will exploit state-of-the-art methods including CRISPR/Cas9 technology in zebrafish, protein biochemistry and quantitative Orbitrap-based mass spectrometry to determine the synaptic targets of enzymes that regulate acetylation levels and to discover how acetylation, in turn, regulates the function of key synaptic proteins. The student will be given in-depth training in these methods and will benefit from collaborations with other groups within the department.

This is a multidisciplinary project between the Collins and Cunliffe labs that will require technology development and cutting-edge methods to generate high-quality quantitative data to understand complex signalling pathways regulating synaptic activity.

References

  • Àlex Bayés, Mark O Collins, Rita Reig-Viader, Gemma Gou, David Goulding, Abril Izquierdo, Jyoti S Choudhary, Richard D Emes & Seth GN Grant. Zebrafish synapse proteome complexity, evolution and ultrastructure. Nature Communications. 2017. Mar 2;8:14613.
  • Wang G, Li S, Gilbert J, Gritton HJ, Wang Z, Li Z, Han X, Selkoe DJ, Man HY. Crucial Roles for SIRT2 and AMPA Receptor Acetylation in Synaptic Plasticity and Memory. Cell Reports. 2017 Aug 8;20(6):1335-1347.

Keywords: Biochemistry, Cell Biology / Development, Genetics, Molecular Biology, Neuroscience/Neurology

Contact information

For informal enquiries about the project or application process, please feel free to contact:

Investigating the links between embryonic laterality and asymmetric morphogenesis of the heart - Emily Noël (COMPETITION FUNDED)

Supervisor: Dr Emily Noël

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Congenital heart diseases occur in around 1% of live births, and are structural defects that arise from improper morphogenesis of the heart during embryonic development. Heart morphogenesis is a highly complex process, drawing on interplay of embryonic laterality cues, tissue rearrangements and cellular migration, and growth and differentiation.

The heart is a highly asymmetric organ. During early stages of development it forms an asymmetrically positioned linear tube that undergoes robust rightward looping to give rise to the looped heart. This stereotypic asymmetric morphogenesis is partly under the control of left-side derived asymmetric gene expression earlier during embryonic development. Importantly, in the absence of laterality signals, the heart can still undergo asymmetric looping morphogenesis, and questions still remain surrounding how this process may be driven, and how heart-extrinsic laterality signals and intrinsic morphogenetic processes are linked. Using transcriptomic analysis we have identified a small number of genes that are upregulated on the left side of the embryo during heart looping, and have been implicated in regulation of the extracellular matrix (ECM).

Furthermore, state of the art light sheet microscopy suggests differential ECM thickness between the left and right sides of the heart during early heart looping. Previous studies have implicated ECM deposition and degradation in earlier stages of heart precursor migration, and later roles in valve formation, however the link between embryonic laterality and asymmetric ECM in the heart is poorly understood.

We are looking for an enthusiastic PhD candidate to explore the link between early left-sided gene expression in the embryo, left-sided ECM deposition in the heart, and heart looping morphogenesis. The main aims of the project will be to:

1. Determine whether left-derived expression of ECM regulators/ECM deposition is under the control of asymmetric laterality cues.

2. Assess the impact of loss of asymmetric ECM deposition upon heart morphogenesis through developing loss-of-function and gain-of-function models.

We use zebrafish as a model organism to study heart development. Zebrafish represent a excellent model with which to study this process due to ease of genetic manipulation, and transparency during embryogenesis allowing us to visualize heart morphogenesis in real time in living embryos. Therefore, the project will provide training in zebrafish transgenesis, genome editing and mutagenesis, as well as state-of-the-art 4D imaging. It will further provide the student with the opportunity to become an expert in standard molecular biology, confocal microscopy, and image analysis. This project should be of particular interest to highly motivated students who have a keen interest in dissecting the molecular links that span embryo-wide signaling, tissue morphogenesis, and cellular responses, and who have an overarching interest in translational research and the genetic basis of congenital diseases.


References

  • Noel et al (2013) A Nodal-independent and tissue-intrinsic mechanism controls heart-looping chirality. Nat Comms
  • Staudt and Stainier (2012) Uncovering the molecular and cellular mechanisms of heart development using the zebrafish. Annu Rev Genet.
  • Rozario and DeSimone (2010) The extracellular matrix in development and morphogenesis: A dynamic view. Dev Bio

Keywords: Cell Biology / Development, Genetics

Contact information

For informal enquiries about the project or application process, please feel free to contact:

Novel genes required for checkpoint control and their role in colo arectal cancer - Carl Smythe

Supervisor 1: Professor Carl Smythe

Supervisor 2: Dr Mark Collins

Funding status: Awaiting funding decision/Possible external funding – find out more on our funding webpage.

Project Description

Background

Healthy aging is frequently affected by the onset of cancers. One in three of us will be affected over our lifetime, and 50% will have significantly reduced lifespan as a consequence. Cell cycle checkpoint genes are often mutated in cancers, while paradoxically, in appropriate circumstances, they may be attractive targets for rational drug design.

Our laboratory was one of the first labs to identify the significance of the Chk1 gene product in checkpoint control and have a significant interest in novel therapeutics and their identification. Colorectal cancer remains a significant challenge, often due to late presentation of symptoms and Chk1 inhibitors are currently in clinical trials for a subset of colorectal cancers. Recently we have undertaken a genome-wide siRNA screen in colorectal cancer cells to identify previously unknown checkpoint pathway genes in order to identify novel potential therapeutic targets. As a result we have identified novel genes whose roles in checkpoint regulation are completely unknown.

Aims

The aims of this project is to gain an understanding of the cellular function of one novel gene, which we have called MiCatS, in cellular checkpoint control. MiCatS-deficient cells appear to be unable to respond to cellular signalling pathways that indicate the presence of replication stress. This project will focus on developing an understanding of how MiCatS interferes with regulatory gene networks controlling genome integrity. Techniques: These will include a range of molecular cell biology techniques, tissue culture, confocal fluorescence microscopy, protein expression and functional characterisation approaches including proteomics, quantitative PCR, siRNA-mediated knockdown of gene expression, and immunoblotting.

References

(1) Feijoo, C., Hall-Jackson, C., Wu, R., Jenkins, D., Gilbert, D., and Smythe, C (2001). Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. The Journal of Cell Biology, 154(5), 913–924.

(2) Bowen, E., (2015). PhD Thesis. “A genomic screen for the identification of novel components of the S phase checkpoint”, University of Sheffield.

Keywords: Cancer / Oncology, Cell Biology / Development, Molecular Biology

Contact information

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Botulinum drugs for migraine treatment - Bazbek Davletov (COMPETITION FUNDED)

Supervisor: Professor Bazbek Davletov

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

This project is open for UK, European and worldwide applicants.

Project Description

Migraine is a common neurological condition, affecting one in five women and one in fifteen men. Current treatments for migraine are often inadequate, and therapeutic advances have been slow. In 2010, quarterly injections of Botulinum neurotoxin type A (Botox®) were approved as a preventative treatment for chronic migraine sufferers. However, only 50% of migraine sufferers report measurable improvement. Botulinum neurotoxin is a potent paralysing agent and thus its efficacy in migraine treatment will always be limited.

Mechanistically, botulinum neurotoxin targets neuronal populations and cleaves intraneuronal SNAP25 protein to cause long-lasting blockade of neurotransmitter release. The project aims to develop a non-paralysing botulinum drug which has improved ability to inhibit release of neuropeptides implicated in migraine. We will use our invented protein-stapling technique to engineer synthetic versions of botulinum neurotoxin which will be analysed in neuronal cells for their targeting and enzymatic efficacies.

The project will involve molecular and cell biological techniques and also in vivo experimentation. Results of this study will help to understand better the biological action of botulinum neurotoxins and most importantly pave the way for new therapeutics to treat a wide range of neurological disorders. This project has the potential to bring about a new approach to treat millions of migraine sufferers.


Keywords: Biochemistry, Biomedical Engineering, Biophysics, Biotechnology, Cell Biology / Development, Genetics, Medical/Clinical Science, Molecular Biology, Neuroscience/Neurology, Pharmacology, Structural Biology, Chemical Engineering, Macromolecular Chemistry, Pharmaceutical Chemistry, Synthetic Chemistry, Biophysics, Nanotechnology

Contact information

For informal enquiries about the project or application process, please feel free to contact:

The role of posttranslational modifications in organisation and function of microtubule cytoskeleton - Natalia Bulgakova (COMPETITION FUNDED)

Supervisor: Dr Natalia Bulgakova

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Correct organization of microtubule cytoskeleton is critical for survival and function of cells in a multicellular organism. Intracellular vesicles and organelles are transported along microtubules to the to places where they are required, and in amounts needed for normal cell functions. We use a simple model organism, the fruit fly Drosophila melanogaster, to study organization and function of microtubule cytoskeleton in epithelial cells.

Recently, we discovered that the organization of microtubule cytoskeleton in epithelial cells is largely influenced by the geometric constraints of the cell. This organization is highly reproducible between cells and genotypes, and is insensitive to perturbations in microtubule dynamics, their number and interactions. We also found that in each cell there are microtubules, which are either acetylated or tyrosinated: the two most common and clinically relevant posttranslational modifications of microtubules.

On the molecular level, acetylation and tyrosination regulate stability and dynamics of microtubules and their interactions with various proteins, for example motor proteins that transport cargo along them. Therefore, these modifications are likely to affect organization and function of microtubule cytoskeleton in a cell. Their incorrect acetylation and tyrosination, however would interfere with cell functions and lead to disease or developmental defects. Indeed, modifications of acetylation and tyrosination profiles of microtubules correlate with cancer progression. However, little is known about how acetylation and tyrosination of microtubules is regulated in an organism, and what are the functions of these modifications on cell and tissue levels.

In collaboration with Dr. Juan Manuel Gomez, University of Cologne, we are currently identifying enzymes that add and remove acetyl and tyrosine groups on microtubules in flies, which will enable understanding roles of these modifications in development and disease. The aim of this project is to understand the role of acetylation and tyrosination in organization of microtubule cytoskeleton and its function using well characterized and reproducible sub-apical microtubule network in Drosophila embryonic epidermis as a model system.

The specific objectives of the project are:

1. To generate Drosophila lines for constitutive and acute inactivation of identified microtubule-modifying enzymes;

2. To determine how these modifications affect organization of microtubule cytoskeleton;

3. To discover roles of these modifications on cell and organism levels.

During the project progression, the student will receive training in a wide range of techniques including molecular biology (generation of Drosophila transgenic and mutant lines using CRISPR/Cas9), state-of-art microscopy (live imaging, super-resolution) and computational approaches.

This project is highly interdisciplinary and will be done in collaboration with Dr. Juan Manuel Gomez, University of Cologne, who will investigate the impact of these modifications in tissue morphogenesis and applied mathematicians from the group of Dr. Lyubov Chumakova, University of Edinburgh, who will develop in silico models of how posttranslational modifications affect organization and function of microtubules in a cell. Altogether, the outcomes of this project will yield fundamental knowledge about regulation of microtubule cytoskeleton, which is relevant to human biology and disease.

Keywords: Cell Biology / Development

References:

  • Gomez, J. M., Chumakova L., Bulgakova N. A., & Brown, N. H. (2016). Microtubule organization is determined by the shape of epithelial cells. Nature Communications, 7, 13172 http://doi.org/10.1038/ncomms13172
  • Bulgakova, N. A., Grigoriev, I., Yap, A. S., Akhmanova, A., & Brown, N. H. (2013). Dynamic microtubules produce an asymmetric E-cadherin-Bazooka complex to maintain segment boundaries. The Journal of Cell Biology, 201(6), 887–901. http://doi.org/10.1083/jcb.201211159

Contact information

For informal enquiries about the project or application process, please feel free to contact:

Elucidating the molecular function of SNAP29 a gene mutated in CEDNIK syndrome - Andrew Peden (COMPETITION FUNDED)

Supervisor: Dr Andrew Peden

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis and keratoderma) syndrome is a rare fatal disease caused by the loss of SNAP29. SNAP29 is member of a family of proteins required for membrane transport and specifically vesicle fusion. We have rshown that SNAP29 is required for the fusion of secretory vesicles with the plasma membrane. Thus, our work suggests that CEDNIK syndrome may be in part be caused by a defect in constitutive secretion.

The aim of this project is to elucidate the molecular details of how SNAP29 functions. Specifically the project will:

1) Determine how SNAP29 is targeted and packaged into post-Golgi vesicles.

2) Identify proteins required for coordinating SNAP29s function. This project will use a combination of super-resolution microscopy, proteomics and CRISPR/Cas9 based techniques.

This project has the potential to elucidate the molecular pathologies which underpin CEDNIK syndrome.

Keywords: Biochemistry, Cell Biology / Development, Genetics, Molecular Biology

References:

  • Gordon D.E., Bond L.M., Sahlender D.A. and Peden A.A. A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells. 2010 Traffic. 11, 1191-1204.
  • Miller S.E., Sahlender D., Graham S., Hoening S., Robinson M.S. Peden A.A and Owen D.J. The Molecular Basis for the Endocytosis of Small R-SNAREs by the Clathrin Adaptor CALM. 2011 Cell 147, 1118-1131.

Contact information

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Elucidating the signalling pathways important for plasma cell biology and antibody secretion - Andrew Peden (Self Funded)

Supervisor 1: Dr Andrew Peden

Supervisor 2: Dr Mark Collins

Funding status: This project is open to self funded students

Project Description

Plasma cells are the antibody secreting cells of the immune system thus are vital for fighting infection. However, dysregulation of plasma cell function is linked to a broad range of disease from Lupus to multiple myeloma. At present, it is not known how plasma cell differentiation and homeostasis is regulated.

The aim of this PhD project is to identify the key signaling pathways critical for this process. To identify these pathways we will use a combination of cutting edge in vitro cell culture models and novel mass-spectrometry based approaches. Once key pathways have been identified, we will manipulate them using chemical and genetic based approaches and determine how this affects plasma cell biology. In the long term, this information may help in the development of novel drugs for regulating plasma cell function in vivo. This project represents an exciting training opportunity where the student will become an expert in advanced proteomics, mechanistic cell biology and immunology.

Keywords: Biochemistry, Bioinformatics, Biotechnology, Cell Biology / Development, Immunology, Molecular Biology, Bioinformatics

Contact information

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Investigating the role of the protease ADAMTS5 in ovarian cancer - Elena Rainero (COMPETITION FUNDED)

Supervisor: Dr Elena Rainero

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

This project is also open to self funded students

Project Description

This project focuses on the interaction between cancer cells and the tumour microenvironment, analysing the role of the extracellular matrix (ECM) modifying enzyme ADAMTS5 in ovarian cancer cell invasive migration. In particular, we will dissect how ADAMTS5 protein is upregulated in ovarian cancer cells and the molecular mechanisms through which the cleavage of the ADAMTS5 substrate versican promotes cancer cell invasion. Ovarian cancer is the most lethal gynaecological malignancy.

Mostly because of late stage diagnosis, the 5-year survival rate is <30%. The major cause of death is associated with the presence of therapy-resistant metastasis. Given the fact that >70% of ovarian cancers are diagnosed at late stage, it is essential to understand what the molecular mechanisms controlling metastasis formation are in order to develop novel strategies for the maintenance of this deadly disease. The tumour microenvironment, including the ECM, has a pivotal role in modulating cancer initiation, progression and metastasis. In particular, Versican (VCAN), a proteoglycan of the lectican family, has been shown to be upregulated in brain tumours, melanomas, osteosarcomas, lymphomas, breast, prostate, colon, lung, pancreatic, endometrial, oral and ovarian cancers and high VCAN expression correlates with reduced overall survival in ovarian cancer patients. VCAN is cleaved by a family of zinc-dependent metalloproteases, A Disintegrin And Metalloproteinase Domain with TromboSpondin type I module (ADAMTS), composed of 19 members of secreted proteases, including ADAMTS5. ADAMTS5 has been shown to increase cell invasive potential in glioblastoma, lung cancer, colon cancer and laryngeal cancer cells. Vesicular trafficking is essential for the polarised distribution of transmembrane receptors and secreted molecules and it is controlled by Rabs, small GTPases of the Ras family.

It is not surprising that alterations in Rab expression and function have been associated with cancer. In particular, epithelial specific Rab25, a member of the Rab11 family, has been shown to promote ovarian cancer cell migration and invasion. Consistent with this, high Rab25 expression correlates with poor prognosis in ovarian cancer. Preliminary data from the lab indicate that Rab25 induces ADAMTS5 expression, and the catalytic activity of ADAMTS5 is required for Rab25-induced cancer cell migration and invasion through 3D environments.

Therefore, we hypothesise that Rab25-expressing ovarian cancer cells upregulate ADAMTS5 expression when in contact with fibroblast-generated ECM. This in turn stimulates their migratory and invasive ability, eventually promoting metastasis formation. This project specifically aims at answering the following questions:

  • How does Rab25 induce ADAMTS5 expression?
  • How does ADAMTS5 promote migration of ovarian cancer cells?
  • Does ADAMTS5 inhibition prevent high grade serous ovarian cancer cell invasion?

Despite the incidence and mortality of ovarian cancer, the mechanisms controlling metastasis are not fully understood. The outcome of this project will deepen our understanding of the interplay between cancer cells and the ECM, highlighting whether ADAMTS5 can represent a novel target for the development of new ovarian cancer therapies.

Keywords: Cancer / Oncology, Cell Biology / Development

Contact information

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Genetic and Drug Screening in DNA Repair Deficient Zebrafish for Novel Targets in the Treatment of Neurological Disease and Cancer - Freek van Eeden (COMPETITION FUNDED)

Supervisor 1: Dr Freek van Eeden

Supervisor 2: Professor Sherif El-Khamisy

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

BBSRC DTP funding available.

Project Description

A cell can experience ~1 million DNA lesions per day from endogenous and exogenous genotoxins. A variety of lesions also result from aberrant replication, or DNA repair itself. DNA lesions threaten essential processes such as transcription and replication and can lead apoptosis and cancer. For example, accumulation of protein-linked DNA breaks (PDBs) cause various neurological diseases, and on the other hand, have been exploited to treat cancer.

Although we know much about DNA-repair pathways from studies in cultured cells, we know little about the extent of functional redundancy at the organismal level. This is important since harnessing this knowledge is rapidly emerging as a powerful approach to treat diseases such as neurodegeneration and cancer. For example, we recently reported, in Nature Neuroscience, a novel mechanism by which PDBs and DNA/RNA hybrids cause motor neuron disease.

Through BBSRC funding and a joint studentship, we established CRISPR mutants in tdp1, brca2, atm, rad52, rad51, which all act in DNA repair, but are mostly viable and often have only mild/no defects as embryos. The objective of the PhD project is to identify backup pathways that can protect the organism if the primary PDB repair pathway is absent. As DNA-repair pathways are often redundant, homozygous mutants provide an excellent background for chemical/genetic modifier screens.

Our primary focus will be on tdp1 mutants, these embryos are - surprisingly- as resistant to DNA damage, as their siblings. We will use CRISPR/CRISPRi technology, and have various chemical libraries available and will screen for defects after induction of DNA damage. We developed an in vivo GFP-reporter system, that uses destruction of a sentinel-repressor to show GFP activation after defective DNA repair. This provides a simple readout in embryos to quantify DNA repair. Importantly, identification of mechanisms behind redundancy may suggest clinical strategies to treat the human disease that results from mutation of tdp1, SCAN1. Moreover, it will provide a platform to stratify cancer patients receiving TDP1 inhibitors currently under development in our labs in collaboration with CRUK technology arm (CRT).

Keywords: Cancer / Oncology, Cell Biology / Development, Genetics, Molecular Biology, Neuroscience/Neurology

Contact information

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Investigating the function of nuclear dystroglycan in prostate cancer - Steve Winder (COMPETITION FUNDED)

Supervisor: Professor Steve Winder

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

This project is also open to self funded students

Project Description

Dystroglycan is an important laminin-binding cell adhesion molecule, crucial for establishment of the basement membrane. Loss of dystroglycan function is symptomatic of almost all adenocarcinomas, with loss of dystroglycan function correlating with cancer progression.

We have established a clear role for dystroglycan in the aetiology of prostate cancer, from growth, through invasion to metastasis. Dystroglycan is particularly important because it mediates the link between inside the cell and the outside extracellular matrix. Dystroglycan is also important because it transmits signals from the outside to the inside of the cell, but also because it can be transported to the nucleus where has two functions, one is to stabilise the nuclear membrane (in a similar way that it does at the cell surface), the other is to control gene expression.

How dystroglycan controls gene expression was not clear, however recent publications that dystroglycan interacts with a regulator of gene expression called YAP, and we hypothesise that dystroglycan bound to YAP could be transported to the nucleus where it has effects on gene regulation. This project will examine the role of dystroglycan and YAP in transport to the nucleus and in controlling gene regulation using normal human prostate cells and prostate cancer cells. The project will use a range of in vitro molecular and cellular approaches complemented by immunofluorescence microscopy and biochemical studies where necessary.

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Medical/Clinical Science, Molecular Biology

Contact information

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The role of nuclear dystroglycan in Duchenne muscular dystrophy - Steve Winder (COMPETITION FUNDED)

Supervisor: Professor Steve Winder

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

This project is also open to self funded students

Project Description

DMD is through to primarily arise due to the lack of dystrophin, which normally acts to stabilise the muscle membrane, leading to breaks in the muscle fibre membrane and muscle fibre death. However, loss of dystrophin also reduces the amounts of other proteins in the muscle membrane, in particular dystroglycan. Dystroglycan is particularly important because it mediates the link between inside the cell and the outside extracellular matrix.

Dystroglycan is also important because it transmits signals from the outside to the inside of the muscle fibre, but also because it can be transported to the nucleus where has two functions, one is to stabilise the nuclear membrane (in a similar way that it does at the cell surface), the other is to control gene expression. How dystroglycan controls gene expression was not clear, however recent publications that dystroglycan interacts with a regulator of gene expression called YAP, and we hypothesise that dystroglycan bound to YAP could be transported to the nucleus where it has effects on gene regulation.

This project will examine the role of dystroglycan and YAP in transport to the nucleus and in controlling gene regulation using myoblast cells from normal human muscle and from muscle from DMD patients. The project will use a range of in vitro molecular and cellular approaches complemented by immunofluorescence microscopy and biochemical studies where necessary.

Keywords: Biochemistry, Cell Biology / Development, Genetics, Molecular Biology

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Mechanisms of cell signalling and coordination of cell polarity in animal development - David Strutt (COMPETITION FUNDED)

Supervisor: Professor David Strutt

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Tissue morphogenesis, repair and regeneration requires cells to communicate and coordinate their behaviours. Major pathways involved are the Wnt/Frizzled and Fat/Dachsous planar polarity pathways that mediate polarised cell signalling in epithelia sheets. Loss of their activity leads to a variety of developmental abnormalities in animal models such as failure of neural tube closure, cleft palates and heart defects, as well as deficits in wound healing and failure to repair kidney damage resulting in polycystic tubules, and is also implicated in cancer metastasis.

The project will focus on understanding molecular mechanisms of Wnt/Frizzled and Fat/Dachsous pathway activity, and in particular how individual proteins adopt polarised distributions with cells, and the signalling mechanisms that propagate this polarity from cell to cell. A molecular genetic approach will be used, taking advantage of the well-established model Drosophila, which provides sophisticated molecular and genetic and cell biological tools. A major focus in the lab is understanding protein dynamics during cell signalling, using techniques such as live imaging (both conventional and super-resolution) and FRAP, and combining this with genetic screens to identify new pathway components and cell biology and biochemical studies of protein behaviours. We combine these experimental studies with computation modelling approaches to aid in experimental design and hypothesis testing.

References

  • Warrington, S.J., Strutt, H., Fisher, K.H. and Strutt, D. (2017) A dual function for Prickle in regulating Frizzled stability during feedback-dependent amplification of planar polarity. Current Biology 27:2784-2797.
  • Strutt, H., Gamage, J. and Strutt, D. (2016) Robust asymmetric localization of planar polarity proteins is associated with organization into signalosome-like domains of variable stoichiometry. Cell Reports 17:2660-2671.
  • Hale, R., Brittle, A.L., Fisher, K.H., Monk, N.A. and Strutt, D. (2015) Cellular interpretation of the long-range gradient of Four-jointed activity in the Drosophila wing. eLife 4, e05789. [PMC4338440]
  • Brittle, A., Thomas, C. and Strutt, D. (2012) Planar polarity specification through the asymmetric subcellular localisation of the atypical cadherins Fat and Dachsous. Current Biology 22: 907-914. [PMC3362735]

Keywords: Cell Biology / Development, Genetics

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Epithelial morphogenesis: coordinating planar polarity and tissue mechanics - David Strutt (COMPETITION FUNDED)

Supervisor 1: Professor David Strutt

Supervisor 2: Dr Alex Fletcher (School of Maths and Statistics)

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

As an organism develops, tissues are shaped and patterned in a coordinated way. The long-standing dogma of developmental biology is that secreted proteins diffuse to form expression gradients throughout tissues thereby providing spatial cues to direct growth, fate and pattern, drawing responses from cells even at some distance from the source. More recently, studies have revealed critical roles for mechanical forces in regulating morphogenesis, however, the interplay between these two systems is poorly understood. With the advent of fast 4D live imaging, combined with genetically encoded fluorescent sensors and sophisticated computational modelling tools, is it now possible to make major advances in understanding epithelial tissue dynamics at a quantitative systems level. The model organism Drosophila provides an ideal system for dissecting mechanisms of morphogenesis in cell sheets, as it is highly amenable to genetic manipulation and has easily accessible simple tissues suitable for live imaging.

The project will integrate cutting-edge genetic tools, advanced 4D fast live imaging and computational modelling in an iterative manner to:
(i) explore how mechanical forces influence patterning and polarity;
(ii) understand how cell division modulates tissue mechanics and coordinated cell polarity; and
(iii) develop mathematical approaches to incorporate pattern and proliferation within existing modelling frameworks for epithelial morphogenesis.

We are looking for an enthusiastic and ambitious student to carry out this interdisciplinary project using both experimental and computational approaches. This project will be suitable for a student with a strong quantitative background (e.g. mathematics, physics, engineering or computer science) who is keen to apply their skills to a biological problem with potentially significant translational importance, or a student with a biological background but a strong interest and some skills in computational approaches. Moreover, the student will be provided with an interdisciplinary training, gaining valuable laboratory experience, particularly in high-level imaging and image analysis, as well as in mathematics and computational modelling.

References

  • Warrington SJ, Strutt H and Strutt D. (2013) The Frizzled-dependent planar polarity pathway locally promotes E-cadherin turnover via recruitment of RhoGEF2. Development 140(5):1045-1054.
  • Strutt H*, Warrington, SJ* and Strutt D. (2011) Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains. Developmental Cell 20: 511-525.
  • Kursawe J, Brodskiy PA, Zartman JJ, Baker RE, Fletcher AG. Capabilities and Limitations of Tissue Size Control Through Passive Mechanical Forces. PLoS Comput Biol. 2015 Dec 29;11(12):e1004679.
  • Tetley RJ, Blanchard GB, Fletcher AG, Adams RJ, Sanson B. Unipolar distributions of junctional Myosin II identify cell stripe boundaries that drive cell intercalation throughout Drosophila axis extension. eLife. 2016 May 16;5:e12094.

Keywords: Biophysics, Cell Biology / Development, Genetics, Biophysics, Applied Mathematics

Contact information

For informal enquiries about the project or application process, please feel free to contact:

Cell plasticity in development and disease - Kyra Campbell (COMPETITION FUNDED)

Supervisor: Dr Kyra Campbell

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

This project is also open to self funded students

Project Description

Although 90% of cancer deaths are caused by metastasis, the underlying pathogenic mechanisms are poorly understood. Cell plasticity is the ability of cells to reversibly change their phenotype and plays a critical role at multiple steps in the complex process of tumour dissemination, which relies on cells undergoing an epithelial-to-mesenchymal transition (EMT), migrating away from the primary tumour and later undergoing a reverse mesenchymal-to-epithelial transition (MET).

While several transcription factors that regulate EMTs have been isolated, how these actually impinge on the cellular responses underlying cell plasticity remains poorly understood. Even less is known about MET: we have so far failed to identify the signals required for its induction, nor do we understand how it occurs at the cell and molecular level. We are looking for a PhD student to work on a project targeted towards identifying the molecular mechanisms underlying epithelial cell plasticity during development and disease.

The project will involve studying how this fundamental property is orchestrated during morphogenesis of the Drosophila midgut, and also in exciting Drosophila cancer models that we have recently generated. This project will combine molecular biology with powerful Drosophila genetics, quantitative image analysis and high-resolution microscopy on our own dedicated multiphoton confocal. It will also involve making targeted Drosophila mutants using CRISPR, and analysing their effects at the subcellular level in fixed and living samples. This is a unique opportunity for you to carry out cutting-edge microscopy, and develop your skills in an exciting multidisciplinary environment.

References

  • Campbell, K. and Casanova, J. (2016). A common framework for EMT and collective cell migration. Development 143, 4291-4300.
  • Campbell, K. and Casanova, J. (2015). A role for E-cadherin in ensuring cohesive migration of a heterogeneous population of non-epithelial cells. Nat Commun 6, 7998.
  • Matorell O, Merlos-Suárez A., Campbell K, Barriga F, Christov C, Miguel-Aliaga I, Batlle E, Casanova J. and Andreu Casali A. (2014). Conserved mechanisms of tumorigenesis in the Drosopihla adult midgut. Plos One.
  • Campbell, K., Whissell, G., Franch-Marro, X., Batlle, E. and Casanova, J. (2011). Specific GATA factors act as conserved inducers of an endodermal-EMT. Dev Cell 21, 1051-1061.

Keywords: Cancer / Oncology, Cell Biology / Development

Contact information

For informal enquiries about the project or application process, please feel free to contact:

Understanding the functional role of adhesion molecules in the synaptogenesis of ribbon synapses in mammalian auditory system - Walter Marcotti (COMPETITION FUNDED)

Supervisor 1: Professor Walter Marcotti

Supervisor 2: Dr Steve Brown (MRC Harwell)

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Hearing is profoundly important in order to perceive information from the outside world and its loss can have a dramatic impact on our daily lives. The perception of hearing depends on the transduction of sound waves into an electrical signal in the auditory organ, the cochlea, which is then transmitted to the brain via auditory afferent neurons. Crucial to this physiological process is the precise, rapid and continuous release of the neurotransmitter glutamate from highly specialized ribbon synapses at the basolateral membrane of sensory inner hair cells (IHCs) located in the mammalian cochlea, which are juxtaposed to the afferent auditory terminals.

We have recently shown that a mutation that affects Neuroplastin caused a failure in the normal synaptogenesis of ribbon synapses in IHCs (Carrott et al 2016). Neuroplastin is a member of the Basigin group of neuronal and synapse-enriched glycoproteins belonging to the immunoglobulin (Ig) superfamily of neural cell adhesion molecules (CAMs), which also includes Basigin and Embigin. All these CAMs are expressed in the mammalian cochlea. Currently, we lack any understanding on the role of Basigin and Embigin on hearing function, as well as the specific involvement of the two encoded Neuroplastin protein isoforms - Neuroplastin-65 and Neuroplastin-55. We will use a combination of well-established and state-of-the-art techniques, together with transgenic mouse models lacking these CAMs to address the hypothesis that this group of proteins is crucial not only for the acquisition, but also for the maintenance of IHC ribbon synapses in hair cells, and as such playing a primary role in hearing. We will also test the hypothesis that these genes are involved age-related hearing loss.

The project will involves a collaboration between the laboratories of Prof Walter Marcotti (Wellcome Trust Senior Investigator, University of Sheffield) and Prof. Steve Brown and Dr Mike Bowl (Director and Project leader, respectively, at the MRC Harwell Institute) who are world-wide experts in hearing and also in the different aspects of this project. You will be trained in a wide-range of in vitro and in vivo techniques, such as patch clamping and 2-photon confocal microscopy, together with the latest techniques in gene expression and mouse genetics. You will also benefit greatly from the wide range of expertise present in the two Institutions through joint lab meeting that are held on a regular basis.

You will complete your PhD having gained a wide range of highly desirable skills, providing a firm foundation for a career in academia or industry. 

Keywords: Biophysics, Cell Biology / Development, Genetics, Neuroscience/Neurology, Biophysics

References:

  • Carrott, L., et al., (2016) Absence of neuroplastin-65 affects synaptogenesis in mouse inner hair cells and causes profound hearing koss. J Neurosci, 36: 222-34.

Contact information

For informal enquiries about the project or application process, please feel free to contact:

Developing tumour treatments by modulating the senescent and secretory phenotypes of cancer-associated fibroblasts using ultrasound - Mark Bass (COMPETITION FUNDED)

Supervisor 1: Dr Mark Bass

Supervisor 2: Dr Daniel Lambert

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Tumours are commonly described as wounds that do not heal. Tumours and chronic wounds comprise dysregulated epithelial cells, senescent fibroblasts, and share similar gene expression profiles. Fibroblast senescence is the major hallmark of chronic wounds, as proliferation defects prevent wound contraction and alters secretion that in turn directs epithelial cell behaviour. Cancer-associated fibroblasts (CAFs) play a similar role in tumour formation. Senescent CAFs promote the growth and metastasis of cancer cells.

Over time, fibroblasts naturally tend towards senescence, which results in a decrease in healing rates and predisposition towards cancer as we age. We recently discovered that low-intensity ultrasound can promote healing in mice with pathological healing defects caused by diabetes or old age, by reversing and protecting fibroblasts from senescence. This PhD will investigate the effect of ultrasound on CAF senescence, leading to the development of new cancer therapies.

Such an application could have two possible impact points:

1) If ultrasound can reduce the senescence of established CAFs, it could be used to abolish the senescence-associated secretory phenotype that supports tumour growth, thereby directly acting as a cancer therapy.

2) One of the greatest limitations of conventional chemotherapy is the collateral damage to surrounding tissue. Therefore, if fibroblasts in proximity to tumours could be protected from stress-induced senescence by ultrasound, conventional chemotherapy could be improved.

Therefore the aims of this project are:

1) To determine whether ultrasound can restore proliferation, metabolism and regulated apoptosis to skin fibroblasts and squamous cell carcinoma-derived CAFs that have already reached either replicative or stress-activated senescence. Such an application would allow treatment of existing cancers.

2) To determine whether application of ultrasound during induction of stress-activated senescence can protect skin fibroblasts and CAFs from senescence, thus allowing the development of future combination therapies.

3) To resolve the effect of ultrasound on the secretory phenotype of senescent fibroblasts. Ultimately, the influence of CAFs over associated tumour cells is more important than the health of the fibroblasts themselves as it will regulate the proliferation and invasion of the tumour itself. This section of the project will assess the effects of senescence and ultrasound on levels of secreted factors, such as IL-6, as well as the effects of fibroblast-conditioned media on tumour cell migration and proliferation.

Therefore this project will involve the investigation of cell proliferation and migration, including both cancer cell migration and invasion analysis in vitro. The project will involve fluorescent techniques including time-lapse microscopy and flow cytometry, as well as ELISA approaches and mass spectrometry to analyse the secretome of treated fibroblasts. The project will place great emphasis on the development of protocols to stimulate live samples with ultrasound, providing great opportunity for innovation under the tutelage of experienced cell biologists and ultrasound experts. Importantly, by running this project in parallel with our ongoing healing studies, there will be many opportunities to meet with clinicians, engineers, and industrial partners thus providing a broad education in research with direct insight into the development of products for clinical application.

Keywords: Biochemistry, Biomedical Engineering, Biotechnology, Cancer / Oncology, Cell Biology / Development, Medical/Biomedical Physics, Medical/Clinical Science, Molecular Biology

References

Roper, Williamson, Bally, Cowell, Brooks, Stephens, Harrison, Bass. (2015)
Ultrasonic stimulation of mouse skin reverses the healing delays in diabetes and aging by activation of Rac1. J Invest Dermatol. 135: 2842
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902130/

Bass, Williamson, Nunan, Humphries, Byron, Morgan, Martin, Humphries. (2011)
A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis. Dev Cell. 21: 681.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3202633/

Kabir, Leigh, Tasena, Mellone, Coletta, Parkinson, Lambert (2016). A miR-335/COX-2/PTEN axis regulates the secretory phenotype of senescent cancer-associated fibroblasts.
Aging 8: 1608
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032686/

Contact information:

For informal enquiries about the project or application process, please feel free to contact

Maintaining effective sensory coding in the face of inter-neuronal variation - Andrew Lin (DIRECTLY FUNDED)

Supervisor 1: Dr Andrew Lin

Supervisor 2: Professor Mikko Juusola and Professor Eleni Vasilaki

Funding status: Directly funded project European/UK students only

Fully funded for 4 years, the studentship covers: (i) a tax-free stipend at the standard Research Council rate (at least £14,553/year for 2018-2019), (ii) research costs, and (iii) tuition fees at the UK/EU rate. The studentship is available to UK and EU students who meet the UK residency requirements, see http://www.bbsrc.ac.uk/documents/studentship-eligibility-pdf/. Students from EU countries who do not meet residency requirements may still be eligible for a fees-only award. See also https://www.whiterose-mechanisticbiology-dtp.ac.uk/

Project Description

When building a brain, you might think neurons should be wired together very precisely and accurately to ensure optimal performance. But nature is never perfect, and developmental variability is inevitable. How can neurons have consistent properties to allow effective sensory coding, in the face of this inherent inter-neuronal variability? This fundamental problem occurs across species, and we will address it in Drosophila, where ~2000 neurons called Kenyon cells encode olfactory associative memories.

To accurately distinguish learned associations for different odours, Kenyon cell population responses to odours must be decorrelated, i.e. different odours activate non-overlapping subsets of Kenyon cells. This inter-odour decorrelation requires Kenyon cells to be roughly equally excitable: if some Kenyon cells are more excitable than others, these same cells tend to dominate all odour responses, which increases overlap between odour representations. Yet recent work shows that Kenyon cells receive extremely variable amounts of excitatory input.

Our computational models suggest that this variability impairs odour decorrelation unless Kenyon cells compensate for variability along one parameter (e.g., amount of excitatory input) with counteracting variability along another parameter (e.g., spiking threshold). In this project, the student will test whether and how such compensatory variability occurs, and will computationally model how it would affect circuit function.

We seek a motivated and creative student with a strong interest in how the brain works. We welcome applications from candidates from a range of backgrounds (from biology to computer science or physics). In carrying out this interdisciplinary project, the student will learn a range of cutting-edge techniques, including multiphoton imaging, patch-clamp electrophysiology, fly genetics, and computational modelling. This research will have broad implications for how neurons develop and maintain the correct electrical and synaptic properties to effectively encode information and carry out behaviourally relevant computations.

Keywords: Bioinformatics, Biophysics, Cell Biology / Development, Genetics, Molecular Biology, Neuroscience/Neurology, Zoology/Animal Science, Applied Mathematics, Data Analysis

References

  • About the model system: Lin, A.C., Bygrave, A.M., de Calignon, A., Lee, T., Miesenböck, G. (2014). Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination. Nature Neuroscience, 17, 559-68.
  • About compensatory variability: Marder, E., and Goaillard, J.-M. (2006). Variability, compensation and homeostasis in neuron and network function. Nat Rev Neurosci 7, 563–574.

Contact information:

For informal enquiries about the project or application process, please feel free to contact

Self Funded Projects

If you are interested in any of the opportunities listed below, please contact the project supervisors directly to enquire about availability.

Investigating the role of the adhesion protein Tensin 3 in breast cancer invasion - Dr Elena Rainero

Supervisor: Dr Elena Rainero

The extracellular matrix (ECM) is a complex network of secreted proteins that, besides providing the scaffolding onto which tissue and organs are organised, is involved in the regulation of a variety of cell function, including survival, growth, migration and differentiation. Moreover, the interaction with the ECM has an important role in controlling tumour formation and metastasis in several cancer types, including breast cancer. Cells interact with the ECM through plasma membrane receptors, including the integrin family, which have a key role in controlling cancer cell invasion and migration. Integrins are not only found at the cell surface, but can be internalised and either be targeted for lysosomal degradation or transported back to the plasma membrane. Interestingly, the way in which integrins are trafficked intracellularly dictates how they control cell migration. My lab focuses on cell-ECM interaction from a novel and exciting angle, analysing the role of the internalisation of ECM components in promoting cancer invasiveness.

My data indicate that invasive breast cancer cells strongly up-regulate ECM uptake, compared to normal mammary epithelial cells, and the integrin-binding protein tensin-3 is required for this. Interestingly, high expression of tensin-3 correlates with poor prognosis in a cohort of breast cancer patients, supporting its role in cancer progression.

The aim of this project is the characterisation of the molecular mechanisms through which tensin-3 controls ECM internalisation and how this impinges on cancer cell invasion, analysing in particular: (1) whether tensin-3 controls ECM endocytosis through the regulation of integrin function; (2) how tensin-3 controls ECM organisation and turnover in 3D environment; (3) what is the role of tensin-3 in cancer cell migration and invasion, in both 2D and 3D settings. To mimic the physiological environment, fibroblast-generated cell-derived matrices and 3D culture systems will be used, coupled with confocal and time-lapse video microscopy.

The outcome of this project will shed new light on the role of ECM trafficking in cancer and will determine whether tensin-3 may represent a target for the development of novel anti-cancer therapeutic strategies.

Keywords: Cancer / Oncology, Cell Biology / Development

For informal enquiries about this project, please contact:

Role of primary cilia in skeletal muscle stem cells and muscle regeneration - Dr Anne-Gaelle Borycki

Supervisor: Dr Anne-Gaelle Borycki

Primary cilia are ancient organelles present at the surface of many cell types in vertebrates. In recent years, they have been shown to play essential roles in relaying sensory and signalling information from the environment to the cell. The importance of primary cilia is underscored by the growing family of diseases associated with defects in cilia function, known as ciliopathies.

Satellite cells are skeletal muscle-specific stem cells responsible for the post-natal growth and repair following injury of skeletal muscles. Satellite cells are normally quiescent, but proliferate and differentiate to repair muscles when they become activated. We have previously shown that quiescent satellite cells exhibit primary cilia, which are rapidly disassembled upon satellite cell activation. Interestingly, we uncovered that in later phases of muscle regeneration, primary cilia re-assemble exclusively at the surface of self-renewing satellite cells and are essential for the maintenance of a stem cell pool in muscles.

This project aims at investigating further the role of primary cilia in satellite cell self-renewal and in muscle regeneration. The project will use a well-established ex-vivo culture system of skeletal muscle fibres as well as in vivo genetic approaches in vertebrates, imaging and molecular biology approaches to investigate whether primary cilia are essential cues for asymmetric cell division and stem cell self-renewal, and to establish the molecular mechanisms controlled by primary cilia in muscle regeneration.

References

  • Jaafar Marican NH, Cruz-Migoni SB, Borycki AG. (2016). Asymmetric Distribution of Primary Cilia Allocates Satellite Cells for Self-Renewal. Stem Cell Reports. 6(6):798-805.

Keywords: Cell Biology / Development, Genetics, Medical/Clinical Science, Molecular Biology

For informal enquiries about this project, please contact:

The role of extra-cellular matrix remodelling in muscle-specific stem cell activity - Dr Anne-Gaelle Borycki

Supervisor: Dr Anne-Gaelle Borycki

Satellite cells are adult skeletal muscle-specific stem cells responsible for post-natal muscle growth and for muscle repair upon injury or disease such as dystrophies. Satellite cells are normally quiescent. However, upon activation, satellite cells exit quiescence, begin proliferating and initiate a myogenic program that culminates with the fusion to the injured muscle fibre and its repair. An important aspect of the repair program is the ability of satellite cells to self-renew, a process that ensures the maintenance of a stem cell pool throughout life allowing the repair of future injuries. Defects in satellite cell self-renewal underlie conditions such as muscular dystrophies and sarcopenia.

It is believed that the control of satellite cell quiescence, proliferation, differentiation and self-renewal is mediated through extrinsic signals produced by the satellite cell niche. We have previously shown that one such signal is delivered by the basal lamina associated with satellite cells, and is required for satellite cell self-renewal. This project aims at investigating further the mechanism of basal lamina control of satellite cell self-renewal. In particular, the project will address the relationship between the satellite cell basal lamina, cell polarity and asymmetric cell division, a process known to underlie self-renewal. The project will use a well-established ex-vivo culture system of skeletal muscle fibres, imaging and molecular biology approaches, combined with mouse genetics to gain a molecular and cellular understanding of satellite cell self-renewal.

Keywords: Cell Biology / Development, Genetics, Medical/Clinical Science, Molecular Biology

For informal enquiries about this project, please contact:

Cellular mechanisms underlying human infection by the intestinal bacterial pathogen enterohaemorrhagic Escherichia coli (EHEC) 0157 - Dr Daniel Humphreys

Supervisor: Dr Daniel Humphreys

Understanding the molecular basis of host-pathogen interactions has never been more important given the health threat posed to humans and farmed food chain animals by bacterial pathogens that continue to develop multidrug resistance. Enterohaemorrhagic Escherichia coli (EHEC) 0157:H7 is an important enteric food-borne pathogen causing life-threatening haemorrhage colitis and haemolytic ureic syndrome in humans. Currently no treatment is available for EHEC infections. Advancing our knowledge of the EHEC virulence mechanisms has the potential to combat disease by speeding the development of anti-infectives and broadening the scope for therapeutic intervention.

To colonise the human intestine EHEC assembles a syringe-like Type 3 Secretion System that injects a cocktail of virulence effector proteins into host cells, which facilitate bacterial colonisation, survival and immune evasion in infected hosts (e.g. see Humphreys et al 2016). The effectors hijack cellular signalling pathways, including those involved in controlling the actin cytoskeleton, vesicle trafficking and cell survival. To combat this disease we need to know the host targets of the EHEC virulence effectors and the mechanisms by which they hijack cellular processes during infection.

We are looking for an enthusiastic and ambitious PhD candidate to investigate interactions between EHEC and mammalian host cells at the Department of Biomedical Science. The student will receive interdisciplinary training in a broad range of techniques, gaining valuable laboratory and research scientist experience, particularly in pathogen and cell biology, and will be an integral part of our research group and a collegiate Department.

The PhD project will combine pathogen genetics and molecular biology to engineer mutant strains of EHEC and recombinant virulence effectors. The recombinant strains will be used to identify pivotal virulence effectors and host cell targets (e.g. of the actin cytoskeleton, vesicle trafficking and/or cell survival) by performing high-throughput infection screens on cultured mammalian cells at the Sheffield RNAi Screening Facility. The screen will be used as a springboard to elucidate novel disease mechanisms by taking advantage of established protein-protein biochemical techniques, mass spectrometry, and cutting-edge fluorescence imaging microscopes at the Wolfson Imaging Light Microscopy Facility (e.g. super-structural resolution, confocal microscopy).

References

  1. Humphreys D. Singh V, Koronakis V. (2016). Inhibition of WAVE Regulatory Complex activation by a bacterial virulence effector counteracts pathogen phagocytosis. Cell Reports, in press.
  2. Humphreys D, Davidson AC, Hume PJ, Makin LE, Koronakis V. (2013) Arf6 coordinates actin assembly through the WAVE complex, a mechanism usurped by Salmonella to invade host cells. Proceedings of the National Academy of Sciences of the United States of America. 110(42):16880-16885. PMID: 24085844
  3. Humphreys D, Davidson AC, Hume PJ, Koronakis V. (2012) Salmonella SopE and host GEF ARNO cooperate to recruit and activate WAVE to trigger bacterial invasion. Cell Host & Microbe 11, 129-39. PMID: 22341462
  4. Smith, K., Humphreys, D., Hume, P.J., and Koronakis, V. (2010) Enteropathogenic Escherichia coli recruits the cellular inositol phosphatase SHIP2 to regulate actin-pedestal formation. Cell Host & Microbe 7, 13-24. PMID: 20114025

Keywords: Biochemistry, Cell Biology / Development, Medical/Clinical Science, Microbiology, Molecular Biology, Pathology

For informal enquiries about this project, please contact:

Mechanisms of cell signalling and coordination of cell polarity in animal development - Professor David Strutt

Supervisor: Professor David Strutt

Tissue morphogenesis, repair and regeneration requires cells to communicate and coordinate their behaviours. Major pathways involved are the Wnt/Frizzled and Fat/Dachsous planar polarity pathways that mediate polarised cell signalling in epithelia sheets. Loss of their activity leads to a variety of developmental abnormalities in animal models such as failure of neural tube closure, cleft palates and heart defects, as well as deficits in wound healing and failure to repair kidney damage resulting in polycystic tubules, and is also implicated in cancer metastasis.

The project will focus on understanding molecular mechanisms of Wnt/Frizzled and Fat/Dachsous pathway activity, and in particular how individual proteins adopt polarised distributions with cells, and the signalling mechanisms that propagate this polarity from cell to cell. A molecular genetic approach will be used, taking advantage of the well-established model Drosophila, which provides sophisticated molecular and genetic and cell biological tools. A major focus in the lab is understanding protein dynamics during cell signalling, using techniques such as live imaging (both conventional and super-resolution) and FRAP, and combining this with genetic screens to identify new pathway components and cell biology and biochemical studies of protein behaviours. We combine these experimental studies with computation modelling approaches to aid in experimental design and hypothesis testing.

References

  • Hale, R., Brittle, A.L., Fisher, K.H., Monk, N.A. and Strutt, D. (2015) Cellular interpretation of the long-range gradient of Four-jointed activity in the Drosophila wing. eLife 4, e05789. [PMC4338440]
  • Strutt, H.*, Searle, E.*, Thomas-MacArthur, V., Brookfield, R. and Strutt, D. (2013) A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins. Development 140: 1693-1702. [PMC3621487]
  • Brittle, A., Thomas, C. and Strutt, D. (2012) Planar polarity specification through the asymmetric subcellular localisation of the atypical cadherins Fat and Dachsous. Current Biology 22: 907-914. [PMC3362735]
  • Strutt, H.*, Warrington, S.J.* and Strutt, D. (2011) Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains. Developmental Cell 20: 511-525. [PMC3094756]

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Genetics

For informal enquiries about this project, please contact:

Cellular mechanisms underlying homeostatic balancing of neuronal excitation and inhibition in vivo - Dr Andrew Lin

Supervisor: Dr Andrew Lin

Neuronal excitation and inhibition are very carefully balanced in the brain, and perturbed excitation/inhibition (E/I) balance has been linked to diseases such as epilepsy, autism and schizophrenia. Maintaining E/I balance within normal bounds depends in part on homeostatic plasticity, in which neurons compensate for deviations in activity levels by adjusting their responsiveness to excitation and inhibition. Although we are starting to understand the molecular mechanisms underlying homeostatic plasticity in reduced preparations, we still know very little about such mechanisms in the intact brain.

We have recently developed a new model system for addressing this question. In the fruit fly Drosophila, Kenyon cells (KCs), the neurons underlying olfactory associative memory, receive excitation from projection neurons as well as feedback inhibition from a single identified neuron. The balance between these two forces maintains sparse odour coding in Kenyon cells, which enhances the odour-specificity of associative memory by reducing overlap between odour representations. Preliminary evidence indicates that Kenyon cells adapt to prolonged disruption of E/I balance, providing a unique opportunity to use the powerful genetic tools of Drosophila to uncover the molecular mechanisms underlying homeostatic plasticity in the intact brain, in a defined circuit that mediates a sophisticated behaviour.

This project will test candidate cellular mechanisms underlying homeostatic compensation. For example, to compensate for insufficient inhibition onto Kenyon cells, excitatory synapses onto Kenyon cells might become weaker or smaller, or Kenyon cells might decrease their input resistance to become intrinsically less excitable. In testing whether these or other mechanisms underlie homeostatic plasticity in vivo, the student will develop skills in a wide range of techniques from fly genetics and confocal microscopy to patch-clamp electrophysiology, two-photon imaging of neural activity, and computational modelling.

References

  • About the model system: Lin, A.C., Bygrave, A.M., de Calignon, A., Lee, T., Miesenböck, G. (2014). Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination. Nature Neuroscience, 17, 559-68.
  • Review about homeostatic plasticity: Davis, G. W. (2013). Homeostatic signaling and the stabilization of neural function. Neuron 80, 718–728.

Keywords: Bioinformatics, Cell Biology / Development, Genetics, Molecular Biology, Neuroscience/Neurology, Bioinformatics

For informal enquiries about this project, please contact:

Epithelial morphogenesis: coordinating planar polarity and tissue mechanics - Professor David Strutt

Supervisors:

As an organism develops, tissues are shaped and patterned in a coordinated way. The long-standing dogma of developmental biology is that secreted proteins diffuse to form expression gradients throughout tissues thereby providing spatial cues to direct growth, fate and pattern, drawing responses from cells even at some distance from the source. More recently, studies have revealed critical roles for mechanical forces in regulating morphogenesis, however, the interplay between these two systems is poorly understood. With the advent of fast 4D live imaging, combined with genetically encoded fluorescent sensors and sophisticated computational modelling tools, is it now possible to make major advances in understanding epithelial tissue dynamics at a quantitative systems level.

The model organism Drosophila provides an ideal system for dissecting mechanisms of morphogenesis in cell sheets, as it is highly amenable to genetic manipulation and has easily accessible simple tissues suitable for live imaging. The project will integrate cutting-edge genetic tools, advanced 4D fast live imaging and computational modelling in an iterative manner to: (i) explore how mechanical forces influence patterning and polarity; (ii) understand how cell division modulates tissue mechanics and coordinated cell polarity; and (iii) develop mathematical approaches to incorporate pattern and proliferation within existing modelling frameworks for epithelial morphogenesis.

We are looking for an enthusiastic and ambitious student to carry out this interdisciplinary project using both experimental and computational approaches. This project will be suitable for a student with a strong quantitative background (e.g. mathematics, physics, engineering or computer science) who is keen to apply their skills to a biological problem with potentially significant translational importance, or a student with a biological background but a strong interest and some skills in computational approaches. Moreover, the student will be provided with an interdisciplinary training, gaining valuable laboratory experience, particularly in high-level imaging and image analysis, as well as in mathematics and computational modelling.

References

  • Warrington SJ, Strutt H and Strutt D. (2013) The Frizzled-dependent planar polarity pathway locally promotes E-cadherin turnover via recruitment of RhoGEF2. Development 140(5):1045-1054.
  • Strutt H*, Warrington, SJ* and Strutt D. (2011) Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains. Developmental Cell 20: 511-525.
  • Kursawe J, Brodskiy PA, Zartman JJ, Baker RE, Fletcher AG. Capabilities and Limitations of Tissue Size Control Through Passive Mechanical Forces. PLoS Comput Biol. 2015 Dec 29;11(12):e1004679.
  • Tetley RJ, Blanchard GB, Fletcher AG, Adams RJ, Sanson B. Unipolar distributions of junctional Myosin II identify cell stripe boundaries that drive cell intercalation throughout Drosophila axis extension. eLife. 2016 May 16;5:e12094.

Keywords: Cell Biology / Development, Genetics, Applied Mathematics

For informal enquiries about this project, please contact:

Host-pathogen interactions of the human fungal pathogen Cryptococcus neoformans - Dr Jason King

Supervisors:

This project will investigate how the fungal pathogen Cryptococcus neoformans interacts with host cells (https://goo.gl/bJaQco). Cryptococcal infection is both a significant opportunistic infection that causes hundreds of thousands of deaths worldwide each year and an excellent opportunity to understand how pathogens avoid killing by the immune system.

Like many opportunistic pathogens Cryptococcus has not evolved specifically to avoid human immune cells. Rather, the normal environmental niche is soil, where its primary concern is capture by predatory amoeba. This project will therefore take a novel approach and use the amoeba Dictyostelium discoideum to investigate what happens to Cryptococcus within a physiologically revelant, model phagocytic cell. This provides an outstanding model system that allows us to genetically manipulate both the host and pathogen, and follow the transit of Cryptococcus through its host in great detail using time lapse fluorescence microscopy. This will allow the successful student to dissect how host and pathogen interact in greater detail than previously possible.

The primary aim of this study is to understand what happens to the human pathogen Cryptococcus neoformans after ingestion by phagocytic cells. This is the first line of defence in the immune system, and the ability of Cryptococcus to manipulate host phagocytes and evade digestion is the major cause of its pathogenicity (goo.gl/IkIf2V). Amazingly Cryptococcus is able to escape macrophages by a process called vomocytosis (goo.gl/BVKzL5). We have shown that Cryptococcus is also able to avoid being killed by Dictyostelium, as well as escape via vomocytosis.

This project with therefore use a combination of genetics, live cell imaging, biochemistry and flow cytometry to identify and characterize how Cryptococcus manipulates its host. This will provide a new level of understanding of cryptococcosis and identify new avenues for therapeutic intervention.

Keywords: Biochemistry, Cell Biology / Development, Genetics, Immunology, Medical/Clinical Science, Microbiology, Molecular Biology

For informal enquiries about this project, please contact:

Please see the King and Johnson lab websites for more details, and recent publications:

The role of the intracellular parts of the Nav1.7 channel in its membrane expression - Dr Mohammed Nassar

Supervisor: Dr Mohammed Nassar

Sensory neurons detect and transmit painful stimuli to the CNS. Inflammation and nerve injury sensitise sensory neurons in vivo which results in a decrease of pain thresholds. This can be due, at least in part, to an enhanced trafficking of voltage gated sodium channels to the plasma membrane which would result in increased excitability of sensory neurons. The VGSC subunit Nav1.7 has been shown to be crucial for pain signaling in mouse and human.

Three genetic pain disorders have been mapped to Nav1.7 in humans; these are primary erthromyalgia, familial rectal pain and complete insensitivity to pain. However and despite its importance in pain signaling, little is known about Nav1.7 transport to nerve terminals, insertion into the membrane and endocytosis from the membrane. Investigation of these processes may lead to new druggable targets for pain relief. Drugs will be directed to inhibit insertion in membrane and increase endocytosis form membrane. Therefore, the aim of this project is to identify the contribution of the intracellular parts of Nav1.7 channel to its membrane expression.

The project will involve using molecular biology methods to generate fusion protein constructs, transfection of DNA into cell lines and primary DRG neurons, immunocytochemistry and Western blotting. Functional effect of transfected fusions on Nav1.7 will be assessed by calcium imaging and patch clamping.

For further details and informal discussion please feel free to contact Dr Nassar.

Keywords: Cell Biology / Development, Molecular Biology, Neuroscience/Neurology

For informal enquiries about this project, please contact:

Elucidating the cellular pathways and machinery involved in epithelial polarisation and cancer using advanced bioimaging - Dr Andrew Peden

Supervisors:

Epithelial polarisation plays an important role in tissue morphogenesis and perturbations in this process can lead to developmental defects and cause cancer. Approximately, 90% of all cancers are derived from epithelial tissue (carcinomas). My group has recently identified a novel vertebrate- and epithelial-specific SNARE, STX19, that is required for the delivery of biosynthetic and endocytic material to the cell surface. Using proteomics and yeast-two-hybrid screening we have identified several novel, STX19 interacting partners that are important players in establishing and maintaining epithelial polarity. It is our hypothesis that STX19 plays a role in the delivery of cell polarity proteins to the cell surface and is required for epithelial polarisation.

To test this hypothesis shRNA will be used to knock down STX19 and its interacting partners in 2 and 3D cell cultures models and epithelial polarisation visualized using structured illumination microscopy. To assess the physiological role of STX19, CRISPR/Cas9 will be also used to generate a STX19 knockout zebrafish and organ development monitored using light sheet microscopy. Using both SIM and light sheet microscopy will allow the processes that drive tissue development to be visualised at an unprecedented spatiotemporal resolution.

This project represents an exciting training opportunity where the student will become an expert in advanced bioimaging, cell biology and animal physiology. This project will not only provide insight into the fundamental cellular processes governing epithelial polarisation and tissue development but also shed light on how changes in these pathways can cause cancer.  

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Molecular Biology

For informal enquiries about this project, please contact:

Obtaining a molecular understanding of antibody secretion - Dr Andrew Peden

Supervisor: Dr Andrew Peden

Plasma cells are the antibody producing cells of the immune system so are vital for fighting infection.  Plasma cells secrete hundreds of millions of antibody molecules per day and this is achieved through a dramatic upregulation of the biosynthetic pathway.

Dysregulation of this process has been implicated in a wide range of diseases from benign autoimmune disorders to metastatic cancer. The aim of this project is to identify novel factors required for regulating antibody secretion and plasma cell function. These factors will be identified using a combination of comparative transcriptomics, proteomics and functional genomics. Genes identified using these methods will increase our fundamental understanding of the secretory pathway and may also be useful for modulating plasma cell function.

The PhD candidate will be trained in techniques such as primary cell culture, proteomics and high-throughput screening.

Keywords: Biochemistry, Bioinformatics, Biotechnology, Cancer / Oncology, Cell Biology / Development, Immunology, Molecular Biology, Bioinformatics

For informal enquiries about this project, please contact:

Cleavage of the protrusion regulator, Vav2, directs cell migration during wound healing - Dr Mark Bass

Supervisor: Dr Mark Bass

Healing delays affect 200,000 UK patients a year, often result in limb amputation, and are caused by defects in the migration of skin fibroblasts.  Upon injury to healthy skin, resident skin fibroblasts are activated and cluster at the wound shoulder where they contract the defect.  Failures in fibroblast migration cause healing delays to the extent that fibroblast senescence is one of the two hallmarks of chronic wounds.  Therefore understanding how migration of fibroblasts towards wound signals is regulated is critical.  The cytoskeletal regulator, Rac1, both drives and guides migration by stimulating localised membrane protrusion. 

A key question is how fibroblasts recognise wound signals and polarise protrusion so that migration to the wound site is efficient.  Rac1 itself is activated by exchange factors such as Vav2, in response to extracellular changes in the wound environment.  We have preliminary evidence that Vav2 mediates the migratory response during healing and that Vav2 is cleaved, immediately after activation, thus ensuring that protrusion signals are transient, making cells responsive to changing migration cues.  This project will identify the Vav2 cleavage site and determine how that cleavage event affects decision making as fibroblasts migrate.

This project will use mass spectrometry to map the site of Vav2 cleavage in response to wound signals, so that cleavage-resistant mutants and pre-cleaved fragments can be generated.  The mutants will be used to investigate the effect of cleavage on Rac1 regulation in cell-based assays and FRET experiments using Rac1 activity probes.  Following characterisation of the effect of cleavage on Rac1, the mechanism will be investigated, thus determining how Vav2 regulation is linked to the extracellular events that occur upon wounding.  Of critical importance will be the investigation of how Vav2 cleavage affects migration.  3D matrices that resemble the skin will be generated and used to investigate the effect of Vav2 cleavage on migration persistence, while micropatterned surfaces will be used to test the effect on decision-making when faced with alternative paths during migration.  Together these experiments will resolve the mechanism by which protrusion is regulated and migration directed during healing.

Finally, the project will link to our ongoing clinical investigations.  We are in the process of developing ultrasonic therapies that restore wound closure in healing-defective animals and patients.  The major limitations to clinical translation of the work are the gaps in our understanding of how Rac1 activation is regulated during healthy healing.  The progress in this PhD project will aid in predicting the healing prognosis of patients, including identification of groups that need and would respond well to intervention.  The effect of the Vav2 cleavage mutants on Rac1 activation in response to ultrasonic stimuli will be tested, thus ensuring that the project connects molecular mechanism to therapeutic outcome and that the student gains understanding of therapeutic development.

References

Roper, Williamson, Bally, Cowell, Brooks, Stephens, Harrison, Bass. (2015) Ultrasonic stimulation of mouse skin reverses the healing delays in diabetes and aging by activation of Rac1. J Invest Dermatol. 135: 2842. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902130/

Williamson, Hammond, Bergen, Roper, Feng, Rendall, Race, Bass. (2014) A coronin-1C/RCC2 complex guides mesenchymal migration by trafficking Rac1 and controlling GEF exposure. J Cell Sci. 127: 4292. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179493/

Bass, Williamson, Nunan, Humphries, Byron, Morgan, Martin, Humphries. (2011) A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis. Dev Cell. 21: 681. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3202633/

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Molecular Biology

For informal enquiries about this project, please contact:

Localisation of Rac1 signals in chronic wound and cancer-associated fibroblasts - Dr Mark Bass

Supervisor: Dr Mark Bass

Healing defects are one of the largest current health challenges.  Chronic leg ulcers affect 200,000 UK patients and cost the NHS £3.1 billion (3% of expenditure) annually, and the rise in linked risk factors such as age and diabetes mean that the challenge is increasing.  Although not fatal, chronic wounds have a huge effect on quality of life as they cause chronic pain and frequently result in amputation of the limb.  Therefore if we are to achieve lifelong health, it is essential that we understand the healing process and identify ways to reverse the defect when healing breaks down.  In healthy individuals, dermal fibroblasts migrate to the wound bed, where they contract to draw the edges of the wound together, but in unhealthy individuals, defects in fibroblast migration are one of the two hallmarks of chronic wounds.

Our laboratory investigates the regulation of migratory signals induced by changes in the extracellular matrix that occur upon wounding.  Directional fibroblast migration requires: a) activation of the protrusion signal, Rac1, at the front of the cell, but suppression at the rear, b) precise temporal regulation of protrusion signals to allow coordination with cell contraction to pull the cell body forward.  We recently reported a Rac1-sequestering molecule, RCC2, and newer data have revealed a link between RCC2 and the transmembrane matrix sensor, syndecan-4.  Genetic disruption of either RCC2 or syndecan-4 perturbs the directionality of fibroblast migration, and both have been demonstrated in vivo.  RCC2-depletion in zebrafish compromises developmental migration, while syndecan-4-knockout in mice retards wound healing.  Both have also been linked to cancer development, and whether this is due to a change in the cancer cells themselves, or cancer-associated fibroblasts remains to be seen.

This project will determine how signalling from syndecan-4 to RCC2 localises Rac1 signalling and thereby directs fibroblast migration.  The composition of the RCC2 complex will be determined by combining syndecan-4 and RCC2 mutants with knockdown of other complex components, identified by mass spectrometry.  The influence of those interactions on Rac1 signalling will be determined using cell-based signalling assays and FRET.  Crucially you will test the effect of the pathways on fibroblast migration through synthetic skin mimics and genuine skin sections.

This project will also link to our ongoing investigations into the use of ultrasound to promote Rac1 activation in cells where matrix-dependent signalling is defective.  We have demonstrated that ultrasound can overturn healing defects in diabetic and syndecan-4-knockout mice and are now testing whether it reverses senescence in both chronic wound and cancer-associated fibroblasts.  The putative role of the syndecan-4/RCC2 pathway in both these instances suggests a link to ultrasound efficacy, meaning that this project will result in clinical impact within a few years.

References

Roper, Williamson, Bally, Cowell, Brooks, Stephens, Harrison, Bass. (2015) Ultrasonic stimulation of mouse skin reverses the healing delays in diabetes and aging by activation of Rac1. J Invest Dermatol. 135: 2842. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902130/

Williamson, Hammond, Bergen, Roper, Feng, Rendall, Race, Bass. (2014) A coronin-1C/RCC2 complex guides mesenchymal migration by trafficking Rac1 and controlling GEF exposure. J Cell Sci. 127: 4292. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179493/

Bass, Roach, Morgan, Mostafavi-Pour, Schoen, Muramatsu, Mayer, Ballestrem, Spatz, Humphries. (2007) Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix. J Cell Biol. 177:527. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885470/

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Molecular Biology

For informal enquiries about this project, please contact:

Integrity of mitochondrial DNA in human pluripotent stem cells and implications for regenerative medicine - Dr Ivana Barbaric

Supervisor: Dr Ivana Barbaric

Human Pluripotent Stem Cells (hPSCs) have the ability to produce cells of all tissues within the body. They can provide a valuable tool for modelling human disease, as well as a potential source of differentiated cells for use in regenerative medicine and drug discovery. In order to fulfil the therapeutic potential of hPSCs it is important to ensure their genome is free of any potentially deleterious mutations that could either impair the function of the hPSC-derived differentiated derivatives or give rise to malignant phenotypes upon transplantation in vivo. Recurrent genetic changes have been noted in the nuclear genome of hPSCs that appear to confer selective growth advantage to the variant cells. Less understood is the appearance and role of mutations in the mitochondrial genome of hPSCs.

This project will focus on investigating the extent of mitochondrial DNA instability, molecular mechanisms that underpin mutation generation and the functional consequences of the mutations on hPSC phenotype and behaviour. Techniques will include a range of molecular and cellular biology methods, including hPSC culture, genetic manipulation of hPSC using CRISPR/Cas9, flow cytometry and live cell imaging.

Keywords: Cell Biology, Development.

For informal enquiries about this project, please contact:

Imaging life beyond the diffraction limit of light - Dr Jarema Malicki

Supervisor: Dr Jarema Malicki

Ever since its invention centuries ago, light microscopy has been hampered by the diffractive properties of light, which limit resolution to ca. 200 nm.  This resolution limit precludes the visualization of many subcellular structures using light microscopy and has been a major obstacle in the imaging of biological processes.  Recently, advances in fluorophore excitation methods and image processing algorithms have overcome this limitation, increasing image resolution by as much as 10 times to about 20 nm.  This new form of imaging is termed super-resolution microscopy.

Super-resolution microscopy opens unprecedented opportunities for imaging biological structures.  We take advantage of this approach to image subcellular structures that regulate intracellular traffic.  We focus on a barrier structure that regulates protein movement between the cell’s cytoplasm and the cilium, a tiny subcellular compartment on the surface of cell.  The cilium is just 250 nm across and so conventional light microscopy cannot be used to visualize its inner architecture.  The inner components of cilia are, however, essential for the function of many cells, tissues, and organs.

We have recently obtained excellent quality super-resolution images in a simple unicellular organism, Tetrahymena.  The purpose of this project is to extend these imaging studies to vertebrate tissues, focusing on the nervous system.  To this end, we will use transgenic lines that express specialized fluorescent proteins suitable for super-resolution imaging in sensory neurons.  This will make it possible to image vital structures of these neurons, such as cilia or synaptic termini, in unprecedented detail and thereby gain insight into the function of these cells.

Keywords: Cell Biology/ Development, Genetics, Neuroscience/Neurology

For informal enquiries about this project, please contact:

or see the Malicki lab website: http://www.malickilab.org/

Developing drugs for long-lasting pain relief - Professor Bazbek Davletov

Supervisor: Professor Bazbek Davletov

This project aims to develop new approach for long-lasting pain relief. Around 12% of adults suffer from severe chronic pain which include cancer, inflammatory and neuropathic pain. Available drugs to treat persistent pain are rarely curative and bring intolerable side effects in a long run. A key feature of our approach is the use of re-targeted botulinum proteases to selectively silence specific types of neurons for months-long periods of time after local injections.

This strategy has evolved from my studies of neuronal communication which universally depends on SNARE proteins. We and others demonstrated that specific cleavage of these proteins can lead to prolonged silencing of neurons with full recovery after several months. My laboratory recently engineered botulinum molecules which have a more selective action. Specifically, several of our molecules target central and sensory neurons but not neuromuscular junctions. This feature makes novel botulinum molecules attractive in treating various chronic neuronal disorders since neuronal silencing can be achieved without side-effects.

We plan to obtain evidence in cell cultures that these botulinum molecules can block release of pain-signalling molecules from sensory neurons and then to test them for their ability to cause long-lasting analgesia in rodent models. Proving the analgesic potential of specific botulinum drugs will be required to translate the benefits of basic research to clinical practice.

References

Mangione AS, Obara I, Maiarú M, Geranton SM, Tassorelli C, Ferrari E, Leese C, Davletov B, Hunt SP. ‘Non-paralytic botulinum molecules for the control of pain. Pain 157:1045-55.

Ferrari E, Gu C, Niranjan D, Restani L, Rasetti-Escargueil C, Obara I, Geranton SM, Arsenault J, Goetze TA, Harper CB, Nguyen TH, Maywood E, O'Brien J, Schiavo G, Wheeler DW, Meunier F, Hastings M, Edwardson JM, Sesardic D, Caleo M, Hunt SP & Davletov B ‘A synthetic self-assembling clostridial chimera for modulation of sensory functions’ Bioconjug Chem 24, 1750−1759.

Arsenault J, Ferrari E, Niranjan D, Cuijpers SAG, Gu C, Vallis Y, O'Brien J & Davletov B. ‘Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells’ J Neurochem 126, 223-33.

Keywords: Biochemistry, Biotechnology, Cell Biology / Development, Molecular Biology, Neuroscience/Neurology, Pharmacology, Veterinary Medicine, Macromolecular Chemistry, Pharmaceutical Chemistry

For informal enquiries about this project, please contact:

Establishment of an in vitro model for cystic fibrosis using organ on chip technology - Dr Kai Erdmann

Supervisor: Dr Kai Erdmann

Cystic fibrosis is a devastating disease affecting the lung and the intestine. Cystic fibrosis is a genetic disease and caused by mutations of the chloride channel CFTR (cystic fibrosis transmembrane conductance regulator). Major phenotypes are increased mucus production in the lung causing extreme breathing difficulties as well as defective signaling in the intestine. One in three thousand newborns are affected.

The goal of this PhD project is the establishment of a state of the art in vitro model for cystic fibrosis to study the disease mechanism in more detail but also to establish a model suitable for high-throughput drug screening. The project will make use of modern organ on chip technology, which allows the functional reconstitution of organ functionality in miniaturized microfluidic devises. Precisely we will apply devices to mimic blood and airflow of the lung allowing a more physiological model system than just 2 or 3D cell cultures.

The aims of this project are
1.) to develop a cellular lung and/or gut model for cystic fibrosis that recapitulates the  phenotype observed in cystic fibrosis.
2.) to incorporate this cellular model into an organ on a chips suitable for mechanistic investigations as well as for high throughput screening
3.) to perform experiments demonstrating suitability of this organ on a chip model for high throughput screening to identify drugs and to analyse the CFTR underlying disease mechanism in more detail.

Techniques: CRISPR-technology, organoids, 3D cell cultures with different mechanical properties, micro-molding using soft-litography, Micropatterning, siRNA-technology, Cell imaging (live confocal spinning disk and super-resolution microscopy, Total-internal reflection microscopy)

References

  • Cutting, G.R. Cystic fibrosis genetics: from molecular understanding to clinical application Nature Reviews Genetics 16, 45-56 (2015)
  • Guggino, W.B. & Stanton, B.A. Mechanisms of disease: New insights into cystic fibrosis: molecular switches that regulate CFTR Nature Reviews Molecular Cell Biology 7, 275-283 (2006)
  • Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science 328, 1662–1668 (2010).

Keywords: Biochemistry, Biotechnology, Cell Biology / Development, Molecular Biology, Neuroscience/Neurology, Pharmacology, Veterinary Medicine, Macromolecular Chemistry, Pharmaceutical Chemistry

For informal enquiries about this project, please contact:

The role of multi-PDZ domain proteins in the regulation of epithelial cell proliferation and cancer - Dr Kai Erdmann

Supervisor: Dr Kai Erdmann

PTPN13 is a highly modular multi-PDZ domain protein composed of an N-terminal KIND (Kinase non catalytic C-lobe) domain, followed by a FERM (Four-point-one/Ezrin/Radixin/Moesin) domain, five PDZ domains, and a tyrosine phosphatase domain assembling a large macromolecular protein complex. There is strong evidence that PTPN13 plays a role in tumor development. In a search for protein tyrosine phosphatases involved in formation of colon carcinoma, it was demonstrated that PTPN13 is mutated in around 10% of colon carcinomas. We have recently identified novel components of the PTPN13 complex. This complex plays an important role in the regulation of cell/cell adhesion and nuclear transcription. However, the precise molecular mechanism, how PTPN13 contributes to tumor development is largely unclear

The goal of this project is to investigate the role of PTPN13 and other multi-PDZ domain proteins in the context of contact inhibition of proliferation, metastasis and tumor invasion. We will apply in vitro cell proliferation, migration and tumor invasion assays as well as state of the art still- and live-imaging microscopy techniques (like spinning disc confocal microscopy and superresolution microscopy) to investigate underlying disease mechanisms.  Furthermore, using in vitro 3-dimensional cell culture systems and modern chip based cell micropatterning we will further analyze the role and molecular mechanism of PTPN13 in epithelial cell proliferation and cell polarization.

References

  •  Hagemann, N., Ackermann, N., Christmann, J., Brier, S, Yu, F., Erdmann, K.S. The serologically defined colon cancer antigen-3 interacts with the protein tyrosine phosphatase PTPN13 and is involved in the regulation of cytokinesis. Oncogene, 32, 4602-4613 (2013)
  • Stenzel, N., Fetzer, C.P., Heumann, R., Erdmann, K.S. PDZ-domain directed basolateral targeting of peripheral membrane proteins in epithelial cells. J. Cell Sci., 122, 3374-3384 (2009)
  • Freiss, G., Chalbos, D. PTPN13/PTPL1: an important regulator of tumor aggressiveness. Anticancer Agents Med Chem. 11, 77-88 (2011)

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Medical/Clinical Science, Molecular Biology

For informal enquiries about this project, please contact:

Developing a novel methotrexate-derived small molecule JAK/STAT pathway inhibitor - Dr Martin Zeidler

Supervisors:

Work in the Zeidler lab has previously identified the anti-folate drug methotrexate (MTX) as a potent JAK/STAT pathway inhibitor. Used at low doses for the treatment of inflammatory and autoimmune conditions such as rheumatoid arthritis, methotrexate is widely used, has a well understood toxicity profile and is a very low cost drug. Given its function as a JAK/STAT inhibitor, the potential exists to repurpose MTX for other diseases associated with inappropriate pathway activity. Such a change in clinical practice has recently been demonstrated and has significant potential to fulfill a significant unmet need.

However, while repurposing MTX has considerable potential, its long term use is also sometimes associated with potentially serious side-effects – most of which are the consequence of its inhibition of dihydrofolate reductase activity (DHFR). MTX side effects range from relatively minor gastro-intestinal discomfort and nausea to potentially serious myelosuppression and foetal loss/defects in early pregnancy. Given these undesirable characteristics, the development of novel MTX-derived compounds which retain their ability to suppress the JAK/STAT pathway, but which no longer inhibit DHFR, have the potential to define a new class of ‘next generation’ JAK/STAT pathway inhibitors.

In this project, cell based assays for JAK/STAT and DHFR activity already established in the Zeidler lab will be combined with novel medicinal chemistry approaches in the Partridge lab. The successful candidate will design and synthesize libraries of novel MTX derivatives designed to explore the chemical space around methotrexate. The candidate will then use these compounds, together with established positive and negative controls, to undertake cell based assays designed to establish the specificity, activity and toxicity of the molecules generated. Lead compounds will be refined using iterative ‘molecular evolution’ approaches and established medicinal chemistry techniques. Ultimately, compounds will be tested in pre-clinical Drosophila and mouse models of human JAK/STAT disease. In the long-term, we aim to engage with the pharmaceutical industry as a prelude to potential human clinical trials.

References:

  • Thomas S, Fisher KH, Snowden JA, Danson SJ, Brown S, Zeidler MP (2015) “Methotrexate is a JAK/STAT pathway inhibitor” PLOSone 10(7) e0130078
  • Palandri, Francesca; Labate, Claudia; Sabattini, Elena; et al. (2016) “Low-dose methotrexate as treatment of myeloproliferative neoplasms: Proof of principle of clinical activity” Am J HEMATOLOGY 91(8) E329-E330

Keywords: Biochemistry, Cancer / Oncology, Medical/Clinical Science, Neuroscience/Neurology, Organic Chemistry, Pharmaceutical Chemistry

For informal enquiries about this project, please contact:

SUMOylation regulation of autophagy induced by ER stress in motor neuron disease - Dr Chun Guo

Supervisor: Dr Chun Guo

Co-supervisor: Professor Matthew Holley

Autophagy is essential for cellular homeostasis and survival. Dysfunctional autophagy has been detected in various neurodegenerative disorders, including Alzheimer's disease (AD) and Amyotrophic lateral sclerosis (ALS)(1), and it is regarded as a suitable target for therapeutic intervention. It leads to stress in the endoplasmic reticulum (ER), which triggers the unfolded protein response (UPR) (2). The UPR has three canonical arms: IRE1α (the inositol-requiring kinase 1 α), PERK and ATF6. Abnormal activation of the IRE1α-XBP1 pathway has been detected in AD and ALS and inhibition of IRE1α activity increases cell viability in ER stress-induced degeneration (3). One function of XBP1 is to suppress neuronal autophagy and its depletion promotes autophagy, enhancing clearance of protein aggregates and ameliorating disease progression in ALS (4). Emerging evidence suggests an important role for protein SUMOylation in UPR, which influences cell survival (5). SUMOylation of XBP1 negatively regulates its function(6), so it is tempting to speculate that it can promote autophagy upon ER stress.

This exciting project will test the hypothesis that protein SUMOylation can reduce disease pathology in inherited forms of ALS by restoring autophagy via the IRE1α-XBP1 pathway. The proposed work will be conducted using a combination of techniques including molecular biology (e.g. cloning, tagging, mutagenesis), protein chemistry (e.g. GST-/His-pulldowns, co-immunoprecipitations (co-IPs), protein purification for assays of SUMOylation and protein interaction, western blotting for the detection of LC3 conversion from LC3-I to LC3-II and degradation of p62), cell culture (clonal cell lines, motor neuronal cell lines and cells derived from ALS animal models or human patients), image analysis of changes in cell/tissue morphology/histology, protein aggregation and ‘autophagic flux’ (e.g. immunocytochemistry, fluorescence and confocal microscopy) and cell viability (e.g. cytochrome c release, caspase activation, MTT, LDH assays).

References:

  1. Nixon, R. A. The role of autophagy in neurodegenerative disease. Nature medicine 19, 983-997, doi:10.1038/nm.3232 (2013).
  2. Scheper, W. & Hoozemans, J. J. The unfolded protein response in neurodegenerative diseases: a neuropathological perspective. Acta neuropathologica 130, 315-331, doi:10.1007/s00401-015-1462-8 (2015).
  3. Ghosh, R. et al. Allosteric inhibition of the IRE1alpha RNase preserves cell viability and function during endoplasmic reticulum stress. Cell 158, 534-548, doi:10.1016/j.cell.2014.07.002 (2014).
  4. Hetz, C. et al. XBP-1 deficiency in the nervous system protects against amyotrophic lateral sclerosis by increasing autophagy. Genes & development 23, 2294-2306, doi:10.1101/gad.1830709 (2009).
  5. Guo, C. & Henley, J. M. Wrestling with stress: roles of protein SUMOylation and deSUMOylation in cell stress response. IUBMB life 66, 71-77, doi:10.1002/iub.1244 (2014).
  6. Chen, H. & Qi, L. SUMO modification regulates the transcriptional activity of XBP1. The Biochemical journal 429, 95-102, doi:10.1042/bj20100193 (2010).

Keywords: Biochemistry, Cell Biology / Development, Genetics, Medical/Clinical Science, Molecular Biology, Neuroscience/Neurology, Pathology, Pharmacology, Statistics

For informal enquiries about this project, please contact:

Regulation of interphase microtubules by actin cytoskeleton - Dr Natalia Bulgakova

Supervisor: Dr Natalia Bulgakova

Intracellular arrangement of microtubule cytoskeleton in interphase cells is diverse across tissues and cell types, ranging from radial patterns to parallel arrays. Aligned microtubules are a hallmark of specialized cell types with non-centrosomal microtubules, such as neuronal and epithelial cells. The microtubule alignment creates a structural scaffold for vectorial transport of different cargos, and therefore is crucial for cell polarity, cell shape, cell migration, and cell-cell communication.

Recently, we found that alignment of microtubules in epithelial cells is largely influenced by two factors. On the one hand, it is achieved by the response of growing microtubules to the geometric constraints of the cell. On the other hand, not all properties of microtubule organisation can be explained by cell geometry. Another type of cytoskeleton, actin, influences arrangement of microtubules, and therefore, their function in epithelial cells. We found that depolymerisation of actin cytoskeleton significantly changes microtubule alignment in Drosophila epithelial cells.

This project will address two related questions:

  1. What is the molecular mechanism of microtubule regulation by actin?
  2. What is the contribution of this regulation to the development of an organism?

During the project progression, the student will receive training in a wide range of techniques including molecular biology, state-of-art microscopy (live imaging, super-resolution) and computational approaches. This project will be done in collaboration with applied mathematicians from the group of Dr. Lyubov Chumakova, University of Edinburg, who will support the findings by mathematical modelling. Altogether, the outcomes of this project will yield fundamental knowledge about regulation of microtubule cytoskeleton, which is relevant to human biology and disease.

References:

  • Gomez, J. M., Chumakova L., Bulgakova N. A., & Brown, N. H. (2016). Microtubule organization is determined by the shape of epithelial cells. Nature Communications, 7, 13172 doi: 10.1038/ncomms13172
  • Bulgakova, N. A., Grigoriev, I., Yap, A. S., Akhmanova, A., & Brown, N. H. (2013). Dynamic microtubules produce an asymmetric E-cadherin-Bazooka complex to maintain segment boundaries. The Journal of Cell Biology, 201(6), 887–901. http://doi.org/10.1083/jcb.201211159
  • Preciado López, M., Huber, F., Grigoriev, I., Steinmetz, M. O., Akhmanova, A., Koenderink, G. H., & Dogterom, M. (2014). Actin-microtubule coordination at growing microtubule ends. Nature Communications, 5, 4778. http://doi.org/10.1038/ncomms5778

Keywords: Cell Biology / Development, Molecular Biology

For informal enquiries about this project, please contact:

JAK/STAT singling and the regulation of the cytoskeleton - Dr Martin Zeidler

Supervisors:

We have previously used the excellent developmental genetics of the Drosophila model organism to investigate the relationship between the JAK/STAT signal transduction pathway and the behaviour of fly blood cells (termed haemocytes) (Bausek & Zeidler 2013, Bina et al 2010). In addition, other researchers have shown that the JAK/STAT pathway is required to rearrange the cytoskeleton of epithelial pole cells to a mesenchymal morphology (EMT) in the developing fly ovary (Silver & Montel 2001). Simultaneously, other studies also suggest a role for human STAT3 in regulating the effectors of EMT in cancers. Taken together, all these studies suggest that the JAK/STAT pathway plays a key role in the regulation of the cytoskeletal changes involved in cell shape changes.

Using technologies developed in Evans lab, we have now have tools available with which to examine the dynamic movements of the haemocyte cytoskeleton in vivo. Using these tools, the responses of cells to stimuli such as nearby wounding or apoptotic cells can be readily examined.

In this project the successful candidate will examine the roles of JAK/STAT pathway signalling and its target genes in vivo. This work will have a particular focus on the haemocytes associated with the third instar larval imaginal discs. In particular, apoptosis of imaginal disc cells requires haemocytes to alter their morphology so as to invade these tissues to remove apoptotic corpses. We now seek to characterise the behaviour of larval haemocytes in response to apoptotic imaginal cells.

Once this ‘wild type’ behaviour has been characterised you will investigate the effect of loss- and gain-of-function mutations in the JAK/STAT pathway (and pathway target genes) on this process and how these genetic manipulations change cellular function and cytoplasmic morphology.

Ultimately this project aims to cast light on the mechanisms underpinning the cellular changes that occur in cancer metastasis.

References:

  • Bausek N, Zeidler MP (2014)“Gα73Β is a downstream effector of JAK/STAT signaling and a regulator of Rho1 in Drosophila hematopoiesis” J Cell Sci 127(1) 101-110
  • Bina S, Wright VM, Fisher KH, Milo M, Zeidler MP. (2010) “Transcriptional targets of
  • Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour
  • Formation” EMBO Rep. 11(3) 201-7
  • Silver DL, Montell DJ. Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell. 2001;107(7):831–41.
  • Wendt, MK, Balanis N, Carlin CR, Schiemann WP. STAT3 and epithelial–mesenchymal transitions in carcinomas JAK-STAT  2014 3(2): e28975

Keywords: Cell Biology / Development, Genetics

For informal enquiries about this project, please contact:

Generating distinct actin networks in a single cytoplasm: Structural studies to determine the impact of actin nucleators on filament structure - Professor Kathryn Ayscough

Supervisors:

A fundamental understanding of how a cell responds to its environment, in order to drive changes to cell physiology, is critical if we are to make relevant and appropriate interventions in the context of disease states. Many years of research have demonstrated that the actin cytoskeleton is a focal point of cell regulation, however there are still large gaps in our understanding of mechanisms governing early steps in actin filament formation and how distinct filament organizations can co-exist in a single cytoplasm. This project will combine molecular biology and biochemistry with advanced electron cryomicroscopy (CryoEM) approaches to gain new structural insights into the process of actin nucleation. It will be supported by world-class labs with expertise in actin biochemistry and cell biology (Ayscough) and in structural analysis of macromolecular complexes using CryoEM (Bullough).
The overall hypothesis that will be tested in this project is that distinct nucleators impose structural constraints facilitating binding of distinct protein subsets to specific filaments.

The Ayscough lab has identified a novel actin filament nucleation and elongation activity in the yeast WASP homologue Las17.  The overall aim of this project is to understand how this nucleation function works and how it interfaces with other nucleating systems in the cell. 

Objectives:

  1. To undertake electron microscopy and reconstruction approaches to determine whether filaments generated in the presence of the WASP family nucleator differ structurally from those generated through other nucleating conditions. 
  2. Use mutagenesis to define key properties required for nucleating and elongating function and determine whether, and how mutations in the nucleator affect F-actin structure.
  3. To investigate how the presence of SH3 domain proteins influence the actin structures generated by the nucleating activity. 

Novelty. The Ayscough lab is pioneering studies in actin nucleation. This project aims to exploit findings emanating from our BBSRC grant and to initiate studies in a more structural direction to generate a greater depth of understanding of current biochemical and cellular findings.

References:

  1. Allwood EG, Tyler JJ, Urbanek AN, Smaczynska-de Rooij II, Ayscough KR. Elucidating Key Motifs Required for Arp2/3-Dependent and Independent Actin Nucleation by Las17/WASP (2016). PLOSOne 11(9):e0163177
  2. Palmer SE, Smaczynska-de Rooij II, Marklew CJ, Allwood EG, Mishra R, Johnson S, Goldberg MW, Ayscough KR. A Dynamin-Actin Interaction Is Required for Vesicle Scission during Endocytosis in Yeast. (2015) Curr Biol. 25:868-78.
  3. Urbanek AN, Smith AP, Allwood EG, Booth WI, Ayscough KR. A novel actin binding motif in Las17/WASP nucleates actin filaments independently of Arp2/3. (2013) Curr Biol 23: 196-203
  4. Jiang S, Wan Q, Krajcikova D, Tang J, Tzokov SB, Barak I, Bullough PA. (2015). Diverse supramolecular structures formed by self-assembling proteins of the Bacillus subtilis spore coat. Mol Microbiol. 97(2):347-59.

Keywords: Biochemistry, Molecular Biology, Structural Biology, Macromolecular Chemistry, Data Analysis

For informal enquiries about this project, please contact:

Bacterial-associated oncogenesis: Investigating the typhoid toxin of the persistent intracellular pathogen Salmonella Typhi in oncogenesis - Dr Daniel Humphreys

Supervisors:

Background

The human intracellular bacterial pathogen Salmonella enterica serovar Typhi (S.Typhi) causes systemic typhoid fever resulting in 27 million cases of disease and 200,000 deaths each year. S.Typhi is a stealth pathogen and a hallmark feature is its ability to also establish persistent chronic infections in the gall bladder where the pathogen is associated with causing cancer. Oncogenesis is exacerbated in humans carrying gene mutations that predispose individuals to gall bladder cancer. This is relevant to public health, as a link between infection and tumour development may result in personalised therapeutic protocols.

How S.Typhi persists in host cells and promotes oncogenesis is not known. S.Typhi initiates infections by injecting virulence proteins into mammalian host cells to direct uptake and replication within intracellular Salmonella-containing vacuoles (SCVs). From its intracellular niche, S.Typhi secretes the typhoid toxin into the extracellular milieu where it enters target host cells. Once inside the cell the toxin traffics to the nucleus where it reprogrammes cell cycle progression, causes DNA damage and manipulates cellular signalling through nuclease and ADP-ribosylase activities. Recent evidence shows that typhoid toxin mediates establishment of persistent infections in animal hosts, and persistence underlies S.Typhi-associated tumour development.
Project

The mechanisms by which typhoid toxin manipulates cells to drive persistent infections are not understood, and the significance of the toxin to S.Typhi’s stealth virulence strategy and bacterial-associated oncogenesis has not been addressed. The PhD project will investigate these research questions using purified toxin derivatives and infection models in combination with the latest advances in molecular cell biology, fluorescence microscopy, and automated high-throughput screening technologies.

The student will join the laboratory of Dr. Daniel Humphreys (first supervisor) at the Department of Biomedical Science (BMS), University of Sheffield. The project will establish assays to study mammalian cells intoxicated with typhoid toxin using molecular, biochemical, genetic and fluorescence imaging approaches. In particular, how the toxin perturbs nuclear functions in cells predisposed to oncogenesis during S.Typhi infection will be examined using high resolution using structural resolution microscopy at the Wolfson Light Microscopy Suite at the BMS.
Having established the project foundations an RNAi screen will be developed with Dr. Steve Brown (third supervisor) at the RNAi Screening Facility, University of Sheffield.

The PhD student will be trained to use robotics and automated microscope systems to perform high-throughput RNAi screens aimed at identifying host genes required for cellular manipulation by the typhoid toxin.

Finally, the student will use Salmonella infection models, intoxication and cell transformation assays to investigate the significance of genes identified in the screen on pathogen persistence and the generation of bacterial-associated oncogenic phenotypes. These project components will be developed in the laboratories of the first supervisor and the second supervisor, Dr. Anjam Kahn, at the University of Newcastle.

To combat typhoid fever we need to understand the hijack mechanisms employed by S.Typhi to cause disease. By bringing together researchers from the University of Sheffield and the University of Newcastle, three areas of expertise will be combined to provide excellent scientific training and exposure to a broad range of techniques in a project of enormous biomedical significance. 

References

  1. Humphreys D*. Singh V, Koronakis V*. (2016). Inhibition of WAVE Regulatory Complex activation by a bacterial virulence effector counteracts pathogen phagocytosis. Cell Reports. 17(3):697-707. PMID: 27732847.
  2. Humphreys D, Davidson AC, Hume PJ, Makin LE, Koronakis V. (2013) Arf6 coordinates actin assembly through the WAVE complex, a mechanism usurped by Salmonella to invade host cells. Proceedings of the National Academy of Sciences of the United States of America. 110(42):16880-16885. PMID: 24085844
  3. Humphreys D, Davidson AC, Hume PJ, Koronakis V. (2012) Salmonella SopE and host GEF ARNO cooperate to recruit and activate WAVE to trigger bacterial invasion. Cell Host & Microbe 11, 129-39. PMID: 22341462
  4. Smith, K., Humphreys, D., Hume, P.J., and Koronakis, V. (2010) Enteropathogenic Escherichia coli recruits the cellular inositol phosphatase SHIP2 to regulate actin-pedestal formation.
    Cell Host & Microbe 7, 13-24. PMID: 20114025

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Genetics, Microbiology, Molecular Biology, Pathology

For informal enquiries about this project, please contact:

Targeting dystroglycan to the nucleus in muscular dystrophy and cancer - Professor Steve Winder

Supervisor: Professor Steve Winder

Background

Dystroglycan is an essential cell adhesion receptor required for early embryonic development. Genetic loss of function gives rise to severe muscular dystrophies with neuronal involvement. Post-translational loss of function also occurs in Duchenne muscular dystrophy and in some cancers. This includes phosphorylation, proteolysis and ubiquitination. Moreover some proteolytic fragments of dystroglycan are targeted to the nucleus where they have effects on transcription. As part of our analysis of dystroglycan post-translational modifications, we identified a lipid modification - palmitoylation of a conserved cysteine residue that could act to anchor bioactive dystroglycan fragments to the membrane, both at the cell surface and in the nucleus.

Aims:

To examine the function of dystroglycan palmitoylation in cellular targeting and in cellular phenotypes associated with muscular dystrophy and cancer.

Techniques:

PCR-based site-directed mutagenesis, and cloning of dystroglycan mutants. Overexpression of dystroglycan mutants in tissue culture cells. Analysis of subcellular distribution of dystroglycan by quantitative immunofluorescence microscopy and cell fractionation. In vitro assays of cell invasion and metastatic growth.

References:

  • Leocadio D, Mitchell A, Winder SJ.
    γ-Secretase Dependent Nuclear Targeting of Dystroglycan.
    J Cell Biochem. 2016 Mar 18. doi: 10.1002/jcb.25537
  • Martínez-Vieyra IA, Vásquez-Limeta A, González-Ramírez R, Morales-Lázaro SL, Mondragón M, Mondragón R, Ortega A, Winder SJ, Cisneros B.
    A role for β-dystroglycan in the organization and structure of the nucleus in myoblasts.
    Biochim Biophys Acta. 2013 1833:698-711
  • G. Mathew, A.Mitchell, J.M.Down, F.C.Hamdy, C.Eaton, D.J.Rosario, S.S.Cross & S.J.Winder (2013)
    Nuclear targeting of dystroglycan promotes the expression of androgen regulated transcription factors in prostate cancer.
    Sci. Rep. 3, 2792.

Keywords: Biochemistry, Cancer / Oncology, Cell Biology / Development, Molecular Biology

For informal enquiries about this project, please contact:

An integrated molecular, cell and proteomic analysis of activity-dependent signalling in neurons - Dr Mark Collins

Supervisor: Dr Mark Collins

Recent proteomic investigations have identified several thousand proteins in biochemically purified synapses and have uncovered multiprotein complexes essential for synapse function. Through these experiments, we have found that many of these synaptic genes are associated with over 100 neurological diseases, highlighting the need for a better a molecular understanding of synapses.

Many genes/proteins have been implicated in different aspects of synaptic plasticity in single gene/low-throughput studies. However, few studies have taken unbiased approaches to measure global synaptic responses at the protein level and therefore it is not clear what the relative significance of a change in expression or phosphorylation of a single protein in the context of the synapse proteome as a whole. The global analysis of early to late signalling events in synaptic stimulation time-course experiments will also add an important temporal dimension to our understanding of signal transduction and processing at synapses.

Furthermore, the extent and importance of receptor-mediated signalling pathway cross-talk is poorly characterised; the molecular dissection of post-translational modification cross-talk at the synapse will be important to understand how signal integration occurs through complex signalling networks. The use of mass spectrometry-based proteomics has great potential to further our understanding of synaptic signalling pathways in an unbiased, quantitative and temporal manner.

This interdisciplinary project will utilise a range of experimental approaches including cell culture, molecular and cell biology, biochemistry and state-of-the-art quantitative mass spectrometry. The student will be given in-depth training in all of these methods and will benefit from collaborations with other groups within the department.

The aims of the project include the following.

  1. Identify protein expression and post-translational modification (e.g. phosphorylation, ubiquitination and palmitoylation) changes that occur after established stimulation paradigms in neuronal cell culture.
  2. Examine temporal features and cross talk of signalling pathways in time course experiments
  3. Characterise the function of activity dependent post-translational modifications on selected disease-relevant genes

References:

  • Coba MP, Pocklington AJ, Collins MO, Kopanitsa MV, Uren RT, Swamy S, Croning MD, Choudhary JS, Grant SG. (2009).
    Neurotransmitters drive combinatorial multistate postsynaptic density networks.
    Science Signaling. Apr 28;2(68):ra19.
  • Ebert DH, Greenberg ME. (2013).
    Activity-dependent neuronal signalling and autism spectrum disorder.
    Nature. Jan 17;493(7432):327-37
  • Cox J, Mann M. (2011).
    Quantitative, high-resolution proteomics for data-driven systems biology.
    Annual Review of Biochemistry 2011;80:273-99.

Keywords: Biochemistry, Cell Biology / Development, Neuroscience/Neurology

For informal enquiries about this project, please contact:

The Role of cilia in processing of visual information - Dr Anton Nikolaev

Supervisors:

Neurons of the vertebrate central nervous system, including these in the hippocampus and the cerebral cortex, are ciliated.  The function of these neuronal cilia remains, however, a mystery.  It is currently believed that central nervous system cilia may contribute to higher brain functions, such as memory and their malfunction may lead to psychiatric disorders, including schizophrenia and autism.

The proposed project will investigate the role of cilia in the processing of visual information in the retina using the zebrafish model. A combination of state-of-the-art techniques, including molecular genetics and 2-photon imaging of neuronal activity, will be used to study how the activity of visual neurons is affected by mutation causing abnormal ciliogenesis.

Keywords: Molecular Biology, Neuroscience/Neurology

For informal enquiries about this project, please contact:

Understanding how memory is recorded in the brain - Dr Anton Nikolaev

Supervisor:

Understanding how memory is stored in the brain is one of the ultimate goals of neuroscience. The cellular basis of memory formation is a change in the synaptic strength during two processes: long term potentiation (LTP) and depression (LTD).  Molecular details of both LTP and LTD are well understood  (reviewed in Citry and Malenka, 2008) but exactly how changes in synaptic strength lead to memory formation is unclear. For example, it is unknown whether memory is stored in a large population of synapses or in changes of individual synapses.

It is also unclear how many bits (or perhaps bytes or even kilobytes?) of information is stored in an individual synapse and how synaptic plasticity is affected during multiple neurological disorders.  To investigate these kinds of problems requires in vivo imaging using optical reporters of synaptic plasticity in individual synapses.  One such reporter, AMPAR-pHluorin, reports trafficking of AMPA receptors during long-term potentiation. It has been used to study molecular details of LTP and LTD in cultured neurons (Ashby et al., 2004), while understanding how LTP and LTD encode memory needs to be performed on behaving animals.

The proposed project aims at using of AMPAR-pHluorin in vivo to understand how visual memory is stored in the zebrafish brain. The zebrafish brain is relatively simple, yet able to memorise a large number of different shapes. The project will utilise state-of-the-art imaging and molecular biology methods to answer the questions outlined above.

Keywords: Neuroscience/Neurology

For informal enquiries about this project, please contact:

Investigating perturbed signalling pathways underlying Motor Neurone Disease - Dr Mark Collins

Supervisor: Dr Mark Collins

Motor Neurone Disease or Amyotrophic lateral sclerosis (ALS) is a disorder that results in fatal paralysis within a few years of symptom onset. Recently, a number of large exome sequencing studies of ALS patients have independently identified several loss of function mutations in the kinase, Tbk1. Repeat expansion in C9ORF72 is a major cause of ALS and results in the formation of aggregates that sequester RNA-binding proteins. Tbk1 phosphorylates optineurin and p62, cargo receptors for recruiting ubiquitinated proteins to the autophagosome for destruction. All three proteins are involved in autophagy and are relevant to the clearance of aggregates that result from C9ORF72 repeat expansion.

This project aims to understand how Tbk1 is involved in disease development and to identify potential targets for therapeutic intervention. This project will exploit a proximity labelling strategy to determine which signaling pathways downstream of Tbk1 are relevant to the formation and accumulation of protein aggregates characteristic of ALS.

This project will utilise a range of experimental approaches including molecular biology (cloning, mutagenesis), cell culture (including patient derived cell lines), protein biochemistry (western blotting, immunoprecipitation, kinase assays), imaging (immunofluorescence/confocal microscopy and state-of-the-art proteomic technologies (high resolution Orbitrap Mass Spectrometry).

References

  • Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia.
    Freischmidt A et al.
    Nat Neurosci. 2015 May;18(5):631-6.
  • Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways.
    Cirulli ET et al.
    Science. 2015 Mar 27;347(6229):1436-41.
  • Filling the Void: Proximity-Based Labeling of Proteins in Living Cells.
    Kim DI, Roux KJ.
    Trends Cell Biol. 2016 Sep 22. pii: S0962-8924(16)30134-9.
  • Decoding signalling networks by mass spectrometry-based proteomics.
    Choudhary C, Mann M.
    Nat Rev Mol Cell Biol. 2010 Jun;11(6):427-39.

Keywords: Biochemistry, Cell Biology / Development, Neuroscience/Neurology

For informal enquiries about this project, please contact:

How do post-translational modifications control the physiology of the human pathogen Campylobacter jejuni? - Dr Mark Collins

Supervisors:

Campylobacter jejuni is the commonest bacterial food-borne pathogen, responsible for an estimated 700,000 cases p.a. of gastroenteritis in the UK alone, mainly from undercooked chicken. The BBSRC priority strategy for eradicating this pathogen from the food chain demands greater understanding of its biology to identify new intervention targets. One important aspect of its metabolism that is poorly understood is the control of protein activity by post-translational modifications (PTMs).

We know that many essential proteins in bacteria are controlled by covalent modifications to their structure. This project seeks to understand the role of these modifications in C. jejuni, particularly phosphorylation, by comparing wild-type and various mutant strains, using state-of-the-art proteomics analyses employing high resolution mass spectrometry and combined with biochemical analysis of protein function. The student will be trained in molecular microbiology including bacterial physiology, mutant construction and global protein expression analysis, and will apply cutting-edge proteomic techniques to an important human pathogen.

This exciting new project will provide an excellent training in a wide range of techniques and has arisen as a result of collaboration between a molecular microbiologist (Dave Kelly) and an expert in post-translational modifications and proteomics (Mark Collins). We seek enthusiastic students who wish to join a team with an established reputation in Campylobacter biology and who wish to become expert in pathogen microbiology, gene cloning and mutagenesis, proteomics and mass spectroscopy as well as protein purification and biochemistry.

Keywords: Biochemistry, Microbiology, Molecular Biology

For informal enquiries about this project, please contact:

New therapeutics for motor neuron diseases - Professor Bazbek Davletov

Supervisor: Professor Bazbek Davletov

Motor neuron diseases such as Parkinson’s and Amyotrophic Lateral Sclerosis (ALS) often lead to autonomic dysfunctions including difficulty swallowing. Dysphagia (swallowing difficulties) is a major risk factor in motor neuron diseases because of lung infections and choking due to saliva production.

Currently, botulinum neurotoxin provides the most reliable, long-lasting partial reduction in salivation. The major obstacle in the use of botulinum neurotoxin stems from its dangerous paralysing properties restricting its application in patients which are already suffering from muscle weakness. We recently developed several non-paralysing botulinum molecules which we need to evaluate in relevant rodent models.

The successful PhD applicant will learn the production of botulinum-based drugs, develop quantitative method for measuring saliva production and investigate the novel therapeutics in rodents. The successful completion of this project will open new avenues for the treatment of motor neuron disease patients ultimately improving the quality of life for people with motor neuron diseases throughout the world.

References

  • Mangione AS, Obara I, Maiarú M, Geranton SM, Tassorelli C, Ferrari E, Leese C, Davletov B, Hunt SP.
    ‘Non-paralytic botulinum molecules for the control of pain.
    Pain 157:1045-55.
  • Ferrari E, Gu C, Niranjan D, Restani L, Rasetti-Escargueil C, Obara I, Geranton SM, Arsenault J, Goetze TA, Harper CB, Nguyen TH, Maywood E, O'Brien J, Schiavo G, Wheeler DW, Meunier F, Hastings M, Edwardson JM, Sesardic D, Caleo M, Hunt SP & Davletov B
    ‘A synthetic self-assembling clostridial chimera for modulation of sensory functions’
    Bioconjug Chem 24, 1750−1759.
  • Arsenault J, Ferrari E, Niranjan D, Cuijpers SAG, Gu C, Vallis Y, O'Brien J & Davletov B
    ‘Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells’
    J Neurochem 126, 223-33.

Keywords: Biochemistry, Biomedical Engineering, Biotechnology, Medical/Clinical Science, Molecular Biology, Neuroscience/Neurology, Pharmacology, Psychology & Psychiatry, Veterinary Medicine

For informal enquiries about this project, please contact:

Rme-6 as an integrator of endocytosis and signalling in cancer cells - Professor Elizabeth Smythe

Supervisor: Professor Elizabeth Smythe

The endocytic pathway regulates intracellular signalling in a variety of ways and when there are defects in this cross-talk, this can give rise to a variety of diseases including cancer. Rab5 is a major regulator of the early endocytic pathway, which cycles between an inactive, cytoplasmic GDP form and an active membrane-associated GTP form. Rab5 guanine nucleotide exchange factors (GEFs) catalyse the conversion of rab5 into its active form where it interacts with a variety of effectors which allow it to perform many different cellular functions, including signalling (Stenmark, 2009).

Our lab is particularly interested in Rme-6 which is a rab5 GEF that integrates signalling and trafficking of receptor tyrosine kinases such as EGFR and Tie2 by modulating flux through the endocytic pathway. Using SILAC mass spectrometry we have identified the interactome of Rme-6 in HeLa cells following EGF stimulation and have identified a number of novel binding partners.

The aim of this PhD project will  be to extend our current understanding of Rme-6 in the integration of endocytosis and signalling through examining the role of these novel binding partners of Rme-6 that interact in a cargo specific manner.

The project will utilise a variety of molecular cell biology approaches, including CRISPr technology to knockout genes of interest as well as knocking in tagged wild-type and mutant proteins, endocytic and signalling assays as well as high resolution light microscopy in fixed and living cells. There will be particular emphasis on the role of Rme-6 in cancer cells.

References

Stenmark, H.
Rab GTPases as coordinators of vesicle traffic.
Nat Rev Mol Cell Biol. 2009 10:513-25.

Semerdjieva S, Shortt B, Maxwell E, Singh S, Fonarev P, Hansen J, Schiavo G, Grant BD, Smythe E.
Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6.
J Cell Biol. 2008 183:499-511.

Keywords: Cell Biology / Development

For informal enquiries about this project, please contact:

Structural analysis of Rme-6, a rab5GEF that integrates endocytosis and signalling - Professor Elizabeth Smythe

Supervisors:

The ras family of small molecular weight GTPases act as molecular switches. In their active GTP conformation, they interact with a variety of effectors to perform a range of physiological functions. Conversion to the GTP conformation is mediated by guanine nucleotide exchange factors (GEFs) while GTPases are inactivated by GTP hydrolysis, facilitated by GTPase activating proteins (GAPs). Ras is the founding member of this family and has many roles in intracellular signalling and mutations in ras can result in cancer.

The rab family of small GTPases regulates many aspects of membrane trafficking and rab5 is considered to be a master regulator of the early endocytic pathway (Stenmark, 2009). Rme-6 is a multidomain protein containing an N-terminal rasGAP domain and a C-terminal rab5 GEF domain connected by a flexible linker. We have evidence that Rme-6 modulates endocytic flux of EGFR resulting in modulation of its downstream signalling by acting as a signalling scaffold.

We have explored the structure of hRme-6 and our recent results have shown that hRme-6 exists as a higher order multimer in vivo and in vitro. Furthermore we have shown its GEF and GAP activities are autoinhibited and require specific spatial activation in cells (2). Recent studies have identified the hinge region as key to regulation of Rme-6 conformation and relief of autoinhibition. We can purify Rme-6 and negative staining and EM analysis have provided a preliminary structure.

The aim of this project will be to refine this structure using cryo-EM approaches. We are particularly existed to understand the role of the hinge region in effecting conformational changes in Rme-6.

References

Stenmark, H.
Rab GTPases as coordinators of vesicle traffic.
Nat Rev Mol Cell Biol. 2009 10:513-25.

Semerdjieva S, Shortt B, Maxwell E, Singh S, Fonarev P, Hansen J, Schiavo G, Grant BD, Smythe E.
Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6.
J Cell Biol. 2008 183:499-511.

Keywords: Biochemistry, Cell Biology / Development, Structural Biology

For informal enquiries about this project, please contact: