Molecular & Cellular Medicine projects

Please try to identify a research project that suits you.  This is to ensure a proper match between your research interests and your prospective supervisor. We do not encourage applications to more than one project however we would encourage informal discussions with different project supervisors to help you decide which project to apply to. Contact details for academic staff can be found on each project description.

Revealing IGFN1 functional roles through in vivo CRISPR/CAS targeting and in vitro mechanical protein unfolding. Supervisors: Dr Gonzalo Blanco & Dr Christoph Baumann

This project will evaluate the implication of IGFN1 in skeletal muscle mechanotransduction and selective autophagy by: 1) Generating an Igfn1 loss of function allele in single adult muscles by CRISPR/CAS mediated genome editing; 2) Measuring the elastic properties of purified IGFN1 by atomic force microscopy. Cytoskeletal protein crosslinkers have been recently placed at the forefront of cellular mechanotransduction. When mechanical strain causes these linkers to unfold, it is proposed that an inducible selective autophagy mechanism is triggered to dispose of damaged proteins. This pathway contributes to protein quality control and is essential for maintaining muscle integrity. However, the role of this pathway in processing the variety of cytoskeletal protein crosslinkers that are required to maintain the sarcomere is not clear. IGFN1 is a large muscle-specific protein conserved in all sequenced mammals with a typical domain composition similar to other proteins that organize and support the sarcomere structure by crosslinking the cytoskeleton. IGFN1 localizes to the Z-disc and is therefore a strong candidate for a mechanotransduction sensor in skeletal muscle. The Igfn1 gene will be targeted using CRISPR/CAS customized vectors previously validated in vitro to generate loss of function mutations in hindlimb muscles of adult mice in vivo. The loss of function effects on muscle structure and function will be characterized in detail by histology, immunofluorescence with autophagy markers and electron microscopy. The lengths obtained by AFM will be related to the physical dimensions of the sarcomeric structures during the cycles of contraction and relaxation. These experiments will provide mechanistic insights into the role of IGFN1 as a structural stabilizer and client of selective autophagy.

Supervisors: Dr Gonzalo Blanco & Dr Christoph Baumann

To discuss your suitability for this project please email: gonzalo.blanco@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

Proteolytic processing of sodium channel β1 subunits. Supervisors: Dr Will Brackenbury & Dr Christoph Baumann

Voltage-gated Na+ channels (VGSCs) are responsible for action potentials in electrically excitable cells, including neurons and muscle cells. VGSCs contain a pore-forming a subunit with one or two auxiliary b subunits. The b subunits regulate channel activity and are unique amongst other ion channel auxiliary subunits because they are also cell adhesion molecules. VGSCs are therefore not only ion channels, but also cell adhesion complexes. In particular, the b1 subunit regulates action potential firing, neurite outgrowth and neuronal migration during central nervous system development. Interestingly, b1 is also expressed in breast cancer cells, where it regulates cellular migration, invasion and metastasis. VGSC b subunits contain cleavage sites for processing by secretases that are involved in Alzheimer’s disease pathology. Secretase activity is also emerging as an important target in breast cancer. However, the functional consequences of proteolytic processing of b1 are not understood. The aim of this project is to test the hypothesis that b1 subunits are cleaved by secretases and that this regulates adhesion, neurite outgrowth, cellular migration and electrical activity. We will use a range of sophisticated ensemble and single-molecule microscopy approaches, e.g. confocal microscopy, TIRF microscopy, FRAP to explore the stoichiometry and cycling of b1 subunits, in neurons and breast cancer cells. We will modulate secretase activity using drugs and by generating b1 mutants in which the cleavage sites have been modified. We will study the functional consequences of b1 processing on gene expression using molecular approaches such as chromatin immunoprecipitation (ChIP). Importantly, we will study the effect of proteolytic processing on cellular migration and channel function using cell migration assays and whole cell patch clamp electrophysiological recording. The project will therefore expose the student to a range of cutting-edge cell biology techniques in labs that are leading in this field. As b1 plays a key role in brain development and in a number of diseases, this project is expected to provide novel mechanistic insights into a potential therapeutic target.

Supervisors: Dr Will Brackenbury & Dr Christoph Baumann

Co-supervisor: Prof Bob White

To discuss your suitability for this project please email: william.brackenbury@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

 

Dissecting the role of microglial LRRK2 kinase in maintaining neuronal function and viability. Supervisors: Dr Sangeeta Chawla & Dr Chris Elliott

Many neurodegenerative disorders, including Parkinson’s, are characterized by a progressive loss of neurons in particular brain regions.  The identification of mutations in leucine-rich repeated kinase 2 (LRRK2) as a major cause of familial autosomal-dominant Parkinson’s has led to intense research on how mutant neuronal LRRK2 might influence neuronal function and viability. Such work has assumed a cell-intrinsic effect of LRRK2 mutations and expressed LRRK2 disease-related mutants specifically in neurons. However, neuronal function and viability are heavily influenced by the microenvironment and cellular interactions with astrocytes and microglia. Microglia are tissue-resident brain macrophages that have recently been implicated in maintaining a healthy brain. A common factor in neurodegenerative disorders is brain inflammation and activation of microglia. Microglia and astrocytes express LRRK2 at reportedly higher levels than neurons, but little is known about how Parkinsonian LRRK2 mutations influence the functions of these non-neuronal cells and what consequences this has for neuronal health and connectivity. This project will use primary cells from rodent brains along with cellular, molecular, biochemical and novel imaging techniques to unravel the cell-specific contributions of wild-type and mutant LRRK2 to neuronal function and viability. We will compare the effects of expressing wild-type and mutant LRRK2 in the 3 individual cell types: microglia, astrocytes and neurons. We will also express LRRK2 in all 3 cells concurrently.  The behavior and function of the cell types will then be studied in mixed co-cultures. We will assess the toxicity of LRRK2 to neurons and assess neuronal parameters that reflect changes in connectivity such as neurite length, neurite branching and synapse numbers. We will examine astrocytic chemokine release and microglial proliferation, motility, cytokine release and phagocytosis.  We will also test the effects of novel LRRK2 pharmacological inhibitors on cell behavior, function and viability. The project will facilitate new biological understanding of the function of normal LRRK2 and the effect of LRRK2 mutations on the physiology of CNS cells.

Supervisors: Dr Sangeeta Chawla & Dr Chris Elliott

To discuss your suitability for this project please email: sangeeta.chawla@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

 

Nuclear matrix support of DNA replication and transcription. Supervisors: Dr Dawn Coverley & Prof Bob White

Visualization of the structure of nuclei has been the main method of cancer diagnosis since light microscopy allowed. However, very little is known about the underlying molecular alterations that give rise to aberrant nuclear architecture, or the effect these have on the organisation of nuclear processes like DNA replication and transcription. Some proteins that are involved in the regulation of DNA replication are immobilized by attachment to the nuclear matrix in normal cells, but not in cancer cells (or in undifferentiated cells). This project will ask whether the same is true for proteins that regulate transcription, in order to test the extent to which published observations (1) can be generalised beyond DNA replication. The project will also look in detail at the CIZ1 protein to identify the proteins that it normally interacts with in the nuclear matrix. Unlike the cytoskeleton, there is currently no consensus on the main protein components of the nuclear matrix, making this approach particularly interesting.

CIZ1 has been implicated in a several common human cancers as well as chronic age-related disorders, and is being developed as the basis for a blood test for cancer (2). Its interaction partners may be similarly useful as markers of nuclear integrity (for application in regenerative medicine strategies or analysis of cancer cells), and might offer new therapeutic drug targets. There are very few molecules that can be used for function-related studies of the nuclear matrix making this work both novel and timely. The project will involve mammalian cell culture and cell cycle synchrony techniques, sub-cellular fractionation to isolate nuclear matrix fractions using a range of techniques, nuclear stability assays to compare the integrity of the nucleus in different cell types, immunofluorescence microscopy and related image quantification techniques, and protein interaction studies including 2D-LC-MS to discover new components of the nuclear matrix.

Supervisors: Dr Dawn Coverley & Prof Bob White

To discuss your suitability for this project please email: dawn.coverley@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

 

Mechanistic insights into the role of MeCP2 in the interpretation of DNA methylation. Supervisors: Dr Darren Goffin & Dr Sangeeta Chawla

Despite identical genetic sequences, neurones are highly divergent with distinct locations, morphology, connectivity, electrophysiology and transcriptional profiles. The epigenome – the patterns of DNA methylation and histone modifications – is ultimately responsible for defining the identity, function and transcriptional architecture of these different cell types, as well as integrating environmental signals into long-lasting changes in gene expression. In particular, DNA methylation plays a fundamental role in modifying DNA-protein interactions, influencing transcriptional states, activity-dependent gene regulation, learning and memory and ageing. Consequently, these alterations in DNA methylation must be properly recognised and interpreted by the cell.

A key player in the recognition and interpretation of DNA methylation is Methyl-CpG Binding Protein 2 (MeCP2). Indeed, mutations within MeCP2 lead to the devastating autism spectrum disorder, Rett syndrome. However, the underlying molecular functions of MeCP2 are poorly understood. We therefore developed novel mouse models that recapitulate disease-associated mutations in MeCP2. We found that these mice manifest Rett syndrome-like phenotypes through a loss of MeCP2 binding to methylated DNA and a concomitant reduction in MeCP2 protein stability (Goffin et al, 2012). Additionally, we found that loss of MeCP2 function leads to alterations in neural circuit function in a cell type-dependent manner (Goffin et al, 2014).

This PhD project will build on these findings and provide new insights into the molecular mechanisms through which MeCP2 recognises and interprets patterns of DNA methylation. Furthermore, this project will examine the cellular and network consequences of preventing the recognition and interpretation of DNA methylation by MeCP2 at the single cell and system levels. This project will employ cutting edge genetic, molecular and electrophysiological approaches to ultimately elucidate the role that DNA methylation plays in regulating proper neuronal function.

Supervisors: Dr Darren Goffin & Dr Sangeeta Chawla

To discuss your suitability for this project please email: darren.goffin@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

The roles of bone marrow stromal-derived thrombopoietin in HSC niche self-renewal. Supervisors: Dr Ian Hitchcock & Dr Mark Coles

Haematopoiesis is a tightly-regulted process of blood cell production that relies on highly-controlled proliferation, self-renewal and differentiation. This occurs predominantly within the bone marrow and is orchestrated by the direct and indirect interactions of many different cell types. One of the most tightly regulated processes involves the maintenance of haematopoietic stem cells (HSCs) in a highly specialised microenvironment known as a HSC niche. Recent findings suggest that bone marrow stromal cells (BMSCs) are critical in regulating the HSC niche through direct cell-cell contact and paracrine signalling. This PhD will use a wide range of cutting-edge techniques to determine which factors produced by stromal cells are important for HSC self-renewal. Techniques involved in this project include a range of cell culture, molecular biology, flow cytometry, 4D imaging and in vivo assays.

This PhD will provide key opportunity to combine state of the art imaging with in vitro and in vivo models to understand a key physiological process in biology, maintenance of the haematopoic stem cell niche.

Supervisors: Dr Ian Hitchcock & Dr Mark Coles

To discuss your suitability for this project please email: ian.hitchcock@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

Structure and Function of HTLV-1 Transcriptional Regulators. Supervisors: Dr Fabiola Martin, Prof Bob White & Dr Fred Antson

HTLV-1 is a virus that incorporates itself into human DNA and can be transmitted through unprotected sex, breastfeeding and contaminated blood transfusions.  An estimated 15-20 million people are infected worldwide, of whom 2-5% develop the aggressive blood malignancy ATLL, which has an average survival time of only nine months.  Another 3% of HTLV-1 carriers develop severe walking disability, which progresses painfully and irreversibly to wheelchair-dependence.  HTLV -1 infection is a neglected disease, for which there is no vaccine or cure.

ATLL is thought to be caused by two proteins produced by the virus in human cells, Tax and HBZ.  We aim to analyse these viral proteins to gain insight into how they function.  This will involve characterising some of the changes they induce in expression of host non-coding RNAs and the mechanism(s) of such changes.  We will also investigate the structures of these key viral proteins, which could be very helpful in understanding how they operate.  Our studies aim to provide detailed insight, at the molecular level, into the properties of the viral proteins that cause malignancy, an important step towards tackling this scourge.   Thorough training will be provided in in biochemistry, structural and molecular biology, geared towards systems of clinical relevance.

Supervisors: Dr Fabiola Martin (Biology/HYMS), Prof Robert White (Biology) & Prof Fred Antson (YSBL)

To discuss your suitability for this project please email: fabiola.martin@hyms.ac.uk

Please read the 'How to apply' tab before submitting your application.

Sweet remedies in regenerative medicine – can complex carbohydrates be used to protect and/or repair the bladder? Supervisors: Prof Jenny Southgate & Prof Jane Thomas-Oates

With age, the healthy functioning of the bladder may deteriorate leading to an array of problems that can affect urinary continence. In particular, the ability of the epithelial lining (the “urothelium”) to provide a urinary barrier that can self-regenerate may become compromised. There is some evidence that instilling the inside of the bladder with complex carbohydrates can improve barrier function, but it is not understood how this works mechanistically – does it provide a physical barrier that enhances the natural sugar coating or glycocalyx? Or does it play a more complex role in urothelial biology – for example by binding and increasing the local availability of growth factors?

This project will use an exciting mix of biology and chemistry approaches to answer these questions. Tissue engineering approaches will be used to generate human urothelium in the laboratory. Quantitative analyses including imaging and electrophysiology will be used to study the effects of applying exogenous carbohydrates on barrier repair and function. Analytical mass spectrometry and glycochemistry approaches will be used to characterize the natural glycocalyx and study the effects of applied carbohydrate therapies in the tissue model. Together this concerted approach will provide new insight into the role of the glycocalyx in urinary barrier function and a platform for the informed development of new therapies in regenerative medicine.

Supervisors: Prof Jenny Southgate (Biology) & Prof Jane Thomas-Oates (Chemistry)

To discuss your suitability for this project please email: jennifer.southgate@york.ac.uk

Please read the 'How to apply' tab before submitting your application.

Targeted screening to identify pathological events in dementia using a Drosophila and Induced Pluripotent Stem Cell Pipeline. Supervisors: Dr Sean Sweeney & Dr Paul Genever

Frontotemporal Dementia (FTD) is a major cause of early onset dementia. Characterised by atrophy of the frontal and temporal lobes with loss in language and social function, FTD incidence has a strong dominant genetic component. Recently FTD has been placed in a neurodegeneration disease spectrum with Motorneuron Disease (MND) suggesting a common pathogenic mechanism. A number of loci for FTD and MND have been mapped. Among these is CHMP2B encoding a subunit of ESCRTIII, a complex required for endosomal function. We have used mutant CHMP2B to perform genetic screens in Drosophila for enhancers and suppressors of an FTD related phenotype in the fly eye. From this point using the Drosophila system we have identified novel conserved molecules and signaling processes contributing to FTD pathogenesis and disrupted neural function (Lu et al., (2013) Mol Cell, 52:264-271, Ahmad et al., (2009) PNAS 106: 12168-71). We wish to confirm our findings in a human system and potentially extrapolate them to other forms of FTD and MND. To do this we intend to generate Induced Pluripotent Stem Cells (iPSCs) in combination with genome editing via Cas9/CRISPR technology to produce FTD/MND affected neurons in vitro. Such disease models can then be tested for confirmation of the pathogenic events and processes identified from the fly system, for the CHMP2B induced form of the disease in addition to other causative loci. The student will work with and gain a working knowledge of Drosophila genetics, neuroscience, iPSC cell culture, Stem Cells, the Cas9/CRISPR system for genome editing, immunocytochemistry and confocal microscopy.

Supervisors: Dr Sean Sweeney & Dr Paul Genever

To discuss your suitability for this project please email: sean.sweeney@york.ac.uk

Please read the 'How to apply' tab before submitting your application.