Cancer Biology & Oncology
Inhibiting cell metabolism to enhance tumour cell death
Supervisors: Dr Salvatore Papa email@example.com & Prof Reuben Tooze
All the cells in our bodies are programmed to die. As they get older, our cells accumulate toxic molecules that make them sick. In response, they eventually break down and die, clearing the way for new, healthy cells to grow. This “programmed cell death” is a natural and essential part of our wellbeing. Every day, billions of cells die like this in order for the whole organism to continue functioning as it is supposed to.
But as with any programme, errors can occur and injured cells that are supposed to die continue to grow and divide. These damaged cells can eventually become malignant and generate tumours. In order to avoid the regular check of programmed cell death, cancer cells reprogram their metabolism so they can cheat death in order to proliferate indefinitely or resist treatment.
Cancer researchers have known for decades that tumours use a faster metabolism compared to normal cells in our body. One classic example of this is that cancer cells increase their consumption of glucose to fuel their rapid growth and strike against programmed cell death.
This means that metabolic reprogramming is pivotal to sustain cancer initiation, growth and progression. As such limiting glucose consumption in cancer cells is becoming an attractive tool for cancer treatments. However, not all cancer cell types are sensitive to the removal of glucose, and even for the cancers that are sensitive, limitation of glucose only slows down the rate of cancer progression. Therefore, the identification of intracellular pathways that regulate metabolic reprogramming of cancer cells is of great interest for possible therapeutic applications.
Our group, in collaboration with Dr Concetta Bubici at Brunel University London, has conducted a series of interdisciplinary studies (Barbarulo et al., Oncogene 2013; Iansante et al., Nature Commun 2015; Lee et al., Front Cell Dev Biol. 2018) to investigate the intracellular mechanisms regulating cell survival (as opposed to apoptosis, a type of programmed cell death). In this project, we will investigate the intracellular pathways regulating the metabolic reprogramming in normal and disease conditions using cell-based techniques and mouse genetics. We will use in-vivo and ex-vivo experimental techniques to study cellular metabolism to understand the functional role of specific genes involved in the regulation of apoptosis in cancer chemoresistance in solid (liver and breast) and haematological (lymphoma and myeloma) cancers, as well as during tissue regeneration and tumour development.
You will gain experience in a broad range of molecular and cell biology techniques including: the powerful ‘gene silencing’ protocols using short-hairpin RNA approaches, lentivirus-mediated shRNA; immunoblotting, immunoprecipitation-complex-based kinase assay, co-immunoprecipitation and pulldown analyses, PCR, gel electrophoresis, ELISA, flow cytometry, drug-testing toxicity, immunohistochemistry, genotyping and in-vivo drug delivery and analyses. All techniques are well established within the laboratory.
1. Lee NCW, Carella MA, Papa S, Bubici C. High Expression of Glycolytic Genes in Cirrhosis Correlates with the Risk of Developing Liver Cancer. Front. Cell Dev. Biol. 6:138. (2018).
2. Verzella D, Bennett J, Fischietti M, Thotakura AK, Recordati C, Pasqualini F, Capece D, Vecchiotti D, D'Andrea D, Di Francesco B, De Maglie M, Begalli F, Tornatore L, Papa S, et al. GADD45β loss ablates innate immunosuppression in cancer. Cancer Res. 78:1275-1292 (2018).
3. Lansante V, Choy PM, Fung SW, Liu Y, Chai J-G, Dyson J, Del Rio A., D’Santos C, Williams R, Chokshi S, Anders RA, Bubici C and Papa S. PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation. Nat Commun, 6:7882 (2015).
The functional characterization of the tumour suppressor gene CSMD1 in breast cancer
Supervisor: Dr Sandra Bell firstname.lastname@example.org
CUB and Sushi multiple domains protein 1 (CSMD1) maps to 8p23, a region deleted in many cancers including breast cancer. Our previous work has established that CSMD1 is an independent prognostic marker in ductal breast cancer, with reduced expression associated with high tumour grade and poor survival1. CSMD1’s function is unknown; however, our data suggests that CSMD1 is involved in a signaling cascade regulating a wide range of cell processes involved in cancer development.
We have already generated shRNA and CRISPR cell lines with reduced CSMD1 expression to investigate the function of CSMD1. Analysis of these cell lines has shown that suppression of CSMD1 expression disrupts cell morphology, in an effect resembling the epithelial-mesenchymal transition (EMT), increases cell proliferation, and enhances cell migration and cell invasion2. We have also established an MCF10A three-dimensional culture model with reduced CSMD1 expression which revealed that loss of CSMD1 expression resulted in the development of larger and more poorly differentiated breast ductal structures2. In addition we have also recently developed an in vivo model with reduced CSMD1 expression. Our aims will be to investigate the role of CSMD1 in metastasis which is the primary cause of cancer-associated mortality. We will study the mechanisms behind the function of CSMD1 in EMT, tumour migration and invasion using our 3D culture and in vivo models.
The PhD student will gain experience of a broad range of molecular and cell biology laboratory techniques including CRISPR, cell culture of 3D models, confocal microscopy, live cell imaging, Gene expression analysis, RT-PCR, pathway analysis, Western blotting, in vivo work, migration and cell invasion assays, immunohistochemistry and immunofluorescence.
1. Kamal M, Shaaban AM, Zhang L, Walker C, Gray S, Thakker N, Toomes C, Speirs V, Bell SM. Loss of CSMD1 expression is associated with high tumour grade and poor survival in invasive ductal breast carcinoma. Breast Cancer Res Treat (2010) 121(3):555-63.
2. Kamal M, Holliday DL, Morrison EE, Speirs V, Toomes C, Bell SM. Loss of CSMD1 expression disrupts mammary duct formation while enhancing proliferation, migration and invasion. Oncology Reports (2017) Jul;38(1):283-292.
3D model of the brain tumour perivascular niche: developing new approaches to target brain tumours
Supervisors: Dr Georgia Mavria email@example.com & Dr Sandra Bell
Aggressive brain tumours have dismal outcomes. Patients with glioblastoma multiforme, or brain metastases arising from tumours in other part of the body such as melanoma and breast cancer, have a median survival of only 14 months when treated with surgery followed by radiation and chemotherapy. This is due to resistance to treatment by cancer stem-like cells (CSCs), cells that have the ability to self-renew and reignite tumour growth following therapy. CSCs preferentially associate with the tumour perivascular niche, and abundance of the niche correlates positively with tumour grade and negatively with patient survival (1, 2). The brain microvasculature and other supportive cells within the niche, including immune cells, pericytes and astrocytes, provide a protective role for CSCs through complex paracrine interactions (1). Understanding these interactions will lead to discovery of new targets, and new and better therapies for brain tumours.
Objectives: You will use the laboratory’s extensive expertise in 3-dimensional organotypic models (3), data obtained from detailed analysis of glioblastoma patient samples, and available patient-derived CSC lines to i) develop a model that recapitulates the key characteristics of the brain tumour perivascular niche, ii) evaluate the response to therapy of CSCs situated within the niche’s protective environment and iii) identify new molecules that promote the growth of CSCs within the niche, and evaluate response to therapy following knockdown their expression.
The project provides an excellent opportunity to train in state-of-the-art cell and molecular biology techniques working within an excellent multidisciplinary environment with medical researchers and clinicians.
1. Charles, N., Holland, E. (2010) The perivascular niche microenvironment in brain tumour progression. Cell cycle 9:3012-21.
2. Ochs, K., et al (2013). Immature mesenchymal stem cell-like pericytes as mediators of immunosuppression in human malignant glioma. J Neuroimmunol. SO165-5728:253-61.
3. Abraham S, Scarcia M, Bagshaw RD, McMahon K, Grant G, Harvey T, Yeo M, Esteves FO, Thygesen HH, Jones PF, Speirs V, Hanby AM, Selby PJ, Lorger M, Dear TN, Pawson T, Marshall CJ and Mavria G (2015). A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis Nat Commun. 6, 7286.
Diagnostics & Pathology
Developing pluripotent stem cell models of inherited retinal diseases
Supervisors: Prof Colin A Johnson firstname.lastname@example.org, Prof C Wälti & Dr K Khan
Background: Inherited retinal dystrophies are a leading cause of blindness and visual loss in the UK working age population. However, despite the widespread diagnostic use of next-generation sequencing, a molecular genetic diagnosis is unavailable for many patients world-wide. Understanding the genetic causes of these conditions enables accurate counselling of patients, improves knowledge of disease pathogenesis and catalyzes the development of novel treatments. To address the current limitations of genetics, this project will develop in vitro cellular modelling of inherited retinal disease in novel microfluidic devices, an approach that is currently under-studied and under-developed. This approach will hugely increase both throughput and the capacity to perform combinatorial assays because existing methods of retinal cell differentiation are costly, laborious and time-consuming.
Environment: We have an existing track-record in induced pluripotent stem cell (iPSC) characterization, differentiation into retinal cell types (retinal pigment epithelium and retinal organoids) and systematic characterization of normal and diseased cellular phenotypes . We have also developed a range microfluidic devices , including for the rapid separation of mesenchymal stromal cells, with minimal manipulation, for autologous cell therapies .
Approach: The project will prototype initial PDMS (polydimethylsiloxane)-based “organ-on-a-chip” devices that enable wide-field microscopy in order to visualize cellular phenotypes. The iPSCs will be differentiated into retinal pigment epithelium following a standard protocol over 21 days . The use of microfluidic devices will minimize culture volumes and cell manipulation, enabling higher throughput and potentially reducing the cost, labour and time that is required for assays of cellular phenotypes. Running several devices in parallel will enable the optimal design to be rapidly chosen for full-scale manufacture. In parallel, we will introduce mutations into normal control iPSCs using CRISPR-Cas9 gene-editing that will model RPE disease in patients. Once the basic design of the chip is optimized for RPE differentiation, we will manufacture multiplexed chips to enable matched sets of combinatorial assays to be performed in parallel (e.g. characterizing a range of cellular phenotypes using a panel of antibodies, reverse genetics screening with a panel of different CRISPR-Cas9 reagents to gene-edit different mutations, testing responses to a panel of drugs or small molecules). The incorporation of transparent indium tin oxide electrodes into the chip will enable transepithelial electrical resistance (TER) assays of cell monolayer integrity in mature RPE. In the longer term, we envisage culture of retinal organoids on chips, and their co-culture with RPE, will enable a more physiological tissue system to be developed. Furthermore, the manufacture of electrodes in the devices will enable us to assay physiological functions such as light-evoked responses.
Outcomes: The project develops methods for in vitro cellular modelling of inherited retinal disease in novel microfluidic devices. This is an approach that provides a more physiological model system for interrogating and understanding human genetic variation in health and disease. It provides unusually broad training research for an excellent graduate who is motivated and proactive in developing a novel area of multi-disciplinary biomedical research.
Techniques used in this project: stem cell culture and differentiation, cellular function assays, confocal microscopy and live cell imaging, CRISPR-Cas9 gene editing (knock-out, knock-in and base editing), microfluidic chip design and manufacture.
1. Buskin A, Zhu L, Chichagova V, Basu B, Mozaffari-Jovin S, …33 others… Grellscheid S-N, Johnson CA, Lako M (2018). Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa. Nat Commun 9:4234. https://rdcu.be/85uN
2. Rimsa R, Smith AJ, Wälti C, Wood CD (2017). A planar surface acoustic wave micropump for closed-loop microfluidics. Appl Phys Lett 111:234102. https://doi.org/10.1063/1.5007701
3. Smith AJ, O'Rorke RD, Kale A, Rimsa R, Tomlinson MJ, Kirkham J, Davies AG, Wälti C, Wood CD (2017). Rapid cell separation with minimal manipulation for autologous cell therapies. Sci Rep 7:41872. https://www.nature.com/articles/srep41872
Gastroenterology & Respiratory Medicine
Gastroesophageal reflux in respiratory disease: pathogenic role and improved management
Supervisors: Prof Lesley A Houghton email@example.com & Dr Daniel Peckham
Gastroesophageal reflux (GER) is associated with many lung diseases, including but not limited to idiopathic pulmonary fibrosis (IPF), non-IPF interstitial lung disease (ILD), chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), non-CF bronchiectasis, asthma and idiopathic chronic cough. Reflux can be substantial, reaching the upper regions of the oesophagus in some patients.Reflux reaching the upper oesophagus in particular, leads to concerns that patients might be aspirating refluxed stomach contents into their lungs, and this might be causing or worsening their lung disease. However, it remains unclear whether these two conditions are actually linked, how they are related, and indeed whether acid suppression, and anti-reflux surgery, has any genuine benefit in these patients.
The University of Leeds, together with Leeds Teaching Hospital Trust, are embarking on an extensive research program to better understand the mechanisms responsible for reflux, particularly proximal reflux, its delayed clearance from the oesophagus (and indeed that of boluses swallowed), along with aspiration in patients with lung diseases, and how this relates to symptoms, disease severity and progression, so that in the future the management of GER may be better “individualised” to the patient.
This project is therefore particularly suited to medically qualified doctors, with a training/interest in respiratory medicine and/or gastroenterology. This project would involve Oesophageal high resolution impedance manometry and analysis using the Chicago Classification, pharyngeal manometry, 24-hr oesophageal pH-impedance, sputum collection, pulmonary function testing
1. Houghton LA, Lee AS, Badri H, DeVault KR, Smith JA. Respiratoy disease and the oesophagus: reflux, reflexes and microaspiration. Nature Reviews Gastroenterology and Hepatology 2016; 13: 445-460.
Genetic studies of corneal endothelial dystrophies and development of alternative treatment options
Supervisor: Dr Manir Ali firstname.lastname@example.org
The cornea is the protective front part of the eye that provides most of the eyes focusing power. The endothelium is a single-cell layer on the inside of the cornea that maintains fluid balance and is required for corneal transparency. The late-onset inherited eye condition, Fuchs endothelial corneal dystrophy (FECD), which is the most common cause of corneal transplantation, affects 5% of people over 40 years old who develop blurred vision as a result of endothelial dysfunction and could lead to blindness. About 70% of cases are caused by a trinucleotide repeat expansion in the intron of TCF4, which causes RNA nuclear foci to develop in the patient’s endothelial cells that leads to cell death. Mendelian mutations in SLC4A11, ZEB1, LOXHD1 and AGBL1 only account for at most 5% of cases leaving about 25% of cases with unidentified mutations.
The primary aim of this project will be to identify the cause of FECD in those cases in whom the mutations have not yet been discovered by initially selecting those with a clear family history. Whole genome sequencing (WGS) and whole exome sequencing (WES) of these cases will identify coding and non-coding variants both in the known FECD genes and in novel genes. These variants will be further studied to provide functional validation of the genetic findings but specific experiments will be determined by the initial findings. Better understanding of the disease mechanisms in light of the molecular diagnosis will support the development of new therapies for this condition.
The techniques used in this PhD will include bioinformatics, analyzing WES and WGS data, PCR, Sanger sequencing, functional validation of variants may include expression profiling, spicing mini- and midi-gene assays, protein structural modelling, RNA sequencing, creating knock-out cell models of putative pathogenic variants by CRISPR-Cas9 genome editing and the study of animal models where available. The search for therapies would include a high throughput small molecule screen with functional readout where that is available.
Investigating the molecular basis of optic nerve degeneration in glaucoma to try and develop a personalized medicine strategy for treatment
Supervisor: Dr Manir Ali email@example.com
The optic nerve is a special sensory nerve that transmits visual impulses from the retina to the brain. Primary Open Angle Glaucoma (POAG) is a chronic, progressive optic neuropathy of multifactorial origin that affects 1 in 10 elderly individuals. The condition is characterised by an elevated intraocular pressure in the eye that causes irreversible damage to the retinal ganglion cells and optic nerve and can remain undiagnosed for many years, only affecting vision when the damage is quite advanced. An early diagnosis before significant vision loss is essential for treating this preventable condition. Glaucoma shows familial aggregation with its prevalence varying among different ethnic groups indicating that genetic factors play a major role in pathogenesis. Mendelian mutations in myocilin, optineurin and WD-repeat-36 of large effect, and at least 20 genetic loci in different genes contributing small effects, only account for at most 5% of cases suggesting that the underlying genetic cause for the majority of cases remains to be discovered.
The primary aim of this project will be to identify POAG patients with defined genetic subtypes enriching for patients with a strong family history. Whole genome sequencing (WGS) and whole exome sequencing (WES) of the familial cases will identify coding and non-coding variants both in the known POAG genes, myocilin, optineurin and WD-repeat-36, and in novel genes. These variants will be further studied on neurological tissue or cells to provide functional validation of the genetic findings but specific experiments will be determined by the initial findings. In each case the better understanding of the disease mechanisms in light of the molecular diagnosis will initiate re-examination of the patients, histories compared and best treatment practise for each subgroup inferred. If successful, this approach may also support the development of new therapies for this complex condition.
The techniques used in this PhD will include PCR, Sanger sequencing, bioinformatics, analyzing WES and WGS data, functional validation of variants may include expression profiling, spicing mini- and midi-gene assays, protein structural modelling, RNA sequencing, creating knock-out cell models of putative pathogenic variants by CRISPR-Cas9 genome editing and the study of animal models where available.
The genetic basis of cognitive impairment in intellectual disability and schizophrenia
Supervisors: Prof Chris Inglehearn firstname.lastname@example.org &
& Dr Manir Ali
Schizophrenia is a chronic mental health condition affecting 1% of human populations, with symptoms that include visual and auditory hallucinations, delusions and disordered thought, leading to disruption of the sense of self. Intellectual disability (ID) is an early-onset neurodevelopmental disorder that impacts on practical well-being and social skills and affects up to 3% of people. There is significant overlap between these common disorders, with families and individuals affected by both and with mechanistic overlap of the environmental and genetic factors involved. Much has been done to identify the underlying genetic causes, but it is evident that the known genes account for only a proportion of susceptibility and much remains to be discovered.
Working with local and international clinical colleagues, we have recruited a series of patients and families affected by either or both of these conditions, and are carrying out research to determine the underlying causes. Current projects include studying a UK family with a Mendelian form of schizophrenia, whole exome sequencing in schizophrenia patients from Qatar where high consanguinity will increase the power to detect recessive susceptibility loci, and examining animal knockout models for a gene in which we found a mutation in a family with intellectual disability.
The student working on this project will use next generation sequencing and bioinformatics to screen new families/cases with schizophrenia/ID for novel causative variants, then a variety of other techniques to determine the mechanisms involved. These could include generating and imaging cellular models by CRISPR gene editing; examining behaviour and pathology of relevant animal models; determining the normal expression pattern of the genes involved and the transcriptomic consequences of variants implicated; carrying out in silico modelling of the effects of specific mutations on the proteins involved; and other analyses as appropriate for the gene, variant and protein involved.
This project is in collaboration with Dr Steve Clapcote in the faculty of Biological Sciences. The appointed student will have the opportunity to learn and carry out bioinformatics analyses of next generation sequencing, and to study the expression patterns and function of newly implicated genes and proteins using a wide range of molecular and cellular biology techniques. Depending on initial genomic findings, work may progress to genome editing and tissue culture, confocal microscopy, live cell imaging, transcript analysis, genome editing and protein modelling studies. This project has the potential to shed new light on human brain development, which is of immense interest both to scientists and to clinicians.
1. Al-Amri A et al (2016) Homozygous single base deletion in TUSC3 causes intellectual disability with developmental delay in an Omani family. Am J Med Genet 170:1826-31.
2. Klaus K et al (2017) The effect of COMT Val158Met and DRD2 C957T polymorphisms on executive function and the impact of early life stress. Brain Behav 7:e00695.
Genome and transcriptome sequencing and functional analysis to find new mutation types in patients with inherited blindness
Supervisor: Prof Chris Inglehearn email@example.com
Human inherited retinal dystrophies (IRDs) result from mutations in over 200 different genes, many of them first implicated by the Leeds Vision Research Group. By comparison with other inherited human conditions they are relatively well understood. Genetic approaches have implicated defects in several well characterised pathways, including phototransduction, cilia formation, pre-mRNA splicing and the recycling of the 11-cis-retinal chromophore. Screening the known genes solves between 50 and 70% of cases, a remarkably high success rate for such a genetically heterogeneous condition. However, it is likely that the common IRD genes have now been found. Further significant improvement in screening success rate is therefore dependent on improving our ability to detect new classes of mutations, present in known genes, which are being missed by existing screening protocols.
This project will use massively parallel sequencing and cutting edge bioinformatics to identify new variants implicated in human retinal diseases, particularly targeting those classes of mutations that are missed or poorly detected by exome screening. To identify these classes of mutation we will use WGS on pre-screened, unsolved cases recruited through Leeds ophthalmic genetics clinics. Cases from other consortia with which the Leeds Vision Research Group collaborates (BRIDGE-SPEED, 100,000 Genomes project, European Retinal Degeneration Consortium) will also be included in the analysis. RNAseq on blood will also be used in selected cases to determine whether this pinpoints a splicing defect or other mechanism in a known IRD gene in a proportion of unsolved cases. Potential causative variants highlighted in these analyses will be subjected to functional testing as appropriate, depending on the nature of the variant and protein and the level of information available in the existing literature.
The appointed student will have the opportunity to learn and carry out bioinformatics analyses of next generation sequencing, both genome and transcriptome, and to study the expression patterns and function of the implicated genes and proteins using a wide range of molecular and cellular biology techniques. Depending on initial findings, work may progress to tissue culture, confocal microscopy, live cell imaging, transcript analysis, genome editing and protein modelling studies.
1. Panagiotou E et al. (2017) Defects in the cell signalling mediator beta-catenin cause retinal vascular condition FEVR, Am J Hum Genetics. 100:960-968.
2. El-Asrag M et al. (2015) Biallelic mutations in the autophagy regulator DRAM2 cause retinal dystrophy with early macular involvement. Am J Hum Genetics. 96:948-54.
How human teeth form and how that process fails in the inherited condition amelogenesis imperfecta
Supervisor: Prof Chris Inglehearn firstname.lastname@example.org
Amelogenesis is the process of enamel formation and is essential for the development of functional teeth. Amelogenesis imperfecta (AI) is a failure of that process. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discoloured, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties and pain. Enamel cannot be regenerated by the human body, making this a life-long, chronic condition. AI affects around 1 in 14,000 in the US, but its prevalence can be much higher in certain populations. To date mutations in 18 genes have been implicated in non-syndromic AI, giving rise to X-linked, recessive and dominant patterns of inheritance in families. However, these genes account for <50% of cases, raising the question of which genes and mutations are implicated in the remaining patients and what function the encoded proteins have in amelogenesis.
This project will screen new families/cases with AI to find novel genes and mutations; test for possible links with more common tooth conditions such as molar incisor hypomineralisation, caries and fluorosis; image the cells which lay down enamel, the ameloblasts, in normal tooth development and in animal models of AI to see how the disease affects the action of these cells; if possible, the project will also model and study the effect of new mutations using genome editing and, where appropriate, carry out in silico modelling of the effects of specific mutations on the proteins involved.
The results of this project will add to the library of AI genes for genetic screening, increasing the success of screening and improving genetic counselling. They will also inform the search for treatment and will improve our understanding of the biology of tooth (and bone) development.
This project will be in collaboration with the Leeds Dental Institute (LDI), School of Medicine, University of Leeds. The appointed student will have the opportunity to learn and carry out bioinformatics analyses of next generation sequencing, and to study the expression patterns and function of newly implicated genes and proteins using a wide range of molecular and cellular biology techniques. Depending on initial genomic findings, work may progress to tissue culture, confocal microscopy, live cell imaging, transcript analysis, genome editing and protein modelling studies.
1. Smith CE et al. (2017) defects in the acid phosphatase ACPT cause recessive hypoplastic amelogenesis imperfecta. Eur J Hum Genetics. doi: 10.1038/ejhg.2017.79
2. Parry DA et al. (2016) Mutations in the pH-Sensing G-protein-Coupled receptor GPR68 cause Amelogenesis Imperfecta. Am J Hum Genetics. 99:984-990
Exploring the genetic contribution to malignant hyperthermia susceptibility and related myopathies
Supervisors: Dr Marie-Anne Shaw email@example.com & Prof Phil Hopkins firstname.lastname@example.org
Malignant hyperthermia (MH) is a potentially lethal form of heat illness that is pharmacogenetically triggered during general anaesthesia in susceptible individuals. The dramatic hyperthermic and hypermetabolic response results from skeletal muscle Ca2+ dysregulation. Although MH is a rare intraoperative event it is invariably fatal unless it is recognised immediately and treated aggressively. MH susceptible patients typically exhibit no overt phenotype unless exposed to any of the commonly used potent inhalational anaesthetics. Whilst the majority of MH susceptible individuals carry a missense variant in the gene coding for the skeletal muscle isoform of the ryanodine receptor, RYR1, a number of other genes are thought to modify the phenotype. In particular we are interested in people who carry a pathogenic variant but do not display the affected phenotype, and phenotypically normal individuals who carry a pathogenic variant.
Currently, MH diagnosis is made using an in vitro contracture test (IVCT) where tissue taken from a skeletal muscle biopsy is exposed to incremental concentrations of halothane and caffeine. The MH Unit, here at Leeds, is the UK reference centre, and holds the largest collection of patient-derived phenotypic data and material worldwide. We have exome sequence for patients and family members. In addition, we have conducted RNAseq analysis on MH susceptible and normal samples, before and after exposure to halothane and caffeine, where we have identified differentially expressed genes that may be directly involved in MH and related myopathies. The initial project will explore the relevance of some of these genes to MH in greater depth. Students will learn about RNAseq analysis and undertake quantitative PCR techniques to measure expression of selected genes. This will be followed by use of bioinformatics tools to identify potential regulators of gene expression. Variants of interest identified through sequence analysis, and tagged regulatory elements, will be genotyped using our extensive sample resource and a ‘case-control’ association approach.
This investigation of variants will be followed by characterisation of selected, predicted myopathic variants in vitro using the CRISPR-Cas9 HDR system. The resultant cell lines will be assayed e.g. for their sensitivity to caffeine, using fluorescent calcium release microscopy. Combinations of variants influencing myopathic phenotypes can be explored, in both human and mouse cultured cells, using a range of techniques. The student will analyse complex phenotypic data in relation to genetic background, whilst working in a group with diverse interests pertaining to Ca2+ regulation.
1. Merritt A, Booms P, Shaw M-A, Miller DM, Daly C, Bilmen JG, Stowell KM, Allen PD, Steele DS, Hopkins PM (2017) Assessing the pathogenicity of RYR1 variants in malignant hyperthermia. Brit. J. Anaesthesia 118, 533-543.
2. Miller DM, Daly C, Aboelsaod EM, Gardner L, Hobson SJ, Riasat K, Shepherd S, Robinson Rl, Bilmen JG, Gupta PK, Shaw M-A, Hopkins PM (2018) Genetic epidemiology of malignant hyperthermia in the United Kingdom. Brit. J. Anaesthesia 121, 944-952.
3. Chang L, Daly C, Miller DM, Allen PD, Boyle JP, Hopkins PM, Shaw M-A (2019) Permeabilized skeletal muscle reveals mitochondrial deficiency in Malignant Hyperthermia susceptible individuals. Brit. J. Anaesthesia 122:613-21.
Oncogenic reprogramming of immune cells
Supervisors: Dr Peter Laslo email@example.com & Dr Fiona Errington-Mais
Under pathogenic challenges, cells of the innate system become epigenetically reprogrammed and establish immune memory. If the myeloid system acquires cellular memory in response to microbial components, could they be similarly reprogrammed by internal pathological signals, namely oncogenes? Could such “oncogene-memory” account for development of drug resistance seen in clinical treatments of myeloid leukaemias?
Chronic myeloid leukaemia (CML) is associated with the BCR-ABL oncogene with 750 patients diagnosed yearly in the UK. Treatment with Imatinib Mesylate (IM), which inhibits the activity of BCR-ABL, has been clinically successful yet ~20% of patients develop drug resistance with imminent death occurring within 12-months.
Given the genetic plasticity of innate immune cells, as well as the clinical observations of drug resistance, it is tempting to speculate that leukaemic myeloid cells can be reprogrammed to become BCR-ABL independent. Definitive proof of such oncogenic programming of the myeloid genome has been lacking.
We established drug resistant clones from the KCL22 cell model; each recapitulating the clinical observations with BCR-ABL activity abolished by IM yet the cells continue to survive. Oncogene-memory was determined by siRNA knockdown approaches whereby targeting of BCR-ABL protein in parental cells induced immediate cell death while drug resistant derivatives continue to grow and survive.
- Molecular characterise the newly reprogrammed gene network that establishes oncogene-memory
- Target specific biological pathways of the defined oncogene-memory (cell cycle, metabolism) in attempts to induce apoptosis thus laying the foundation for future generation of novel therapies.
The PhD project will employ a systems-biology approach (genome wide expression analysis, bio-informatics and shRNA technology) with the specific aims to (i) identify regulatory factors whose expression is dysregulated as a direct consequence of BCR-ABL activity and (ii) attempt to rescue the developmental block by restoring the functional activity of these dysregulated genes.
The PhD student will gain experience in a broad range of molecular and cell biology techniques including standard recombinant DNA procedures, gene expression profiling, micro-array analysis, bio-informatics, shRNA and general tissue culture practice with culturing of both primary and established cell lines.
1. Laslo P and Stopka T. Transcriptional and epigenetic regulation in the development of myeloid cells: normal and diseased myelopoiesis. Book Chapter. Epigenetics and Human Health Springer, 2014
2. Corbin, A. S. et al. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. The Journal of clinical investigation 121, 396-409, (2011).
3. Cilloni, D. & Saglio, G. Molecular pathways: BCR-ABL. Clin Cancer Res 18, 930-937, (2012).
Enhancing reovirus immunotherapy with eicosapentaenoic acid (EPA)
Oncolytic viruses (OVs) are viruses that 1) replicate preferentially in cancer cells, causing cell death, and 2) induce anti-tumour immunity, through activation of both innate and adaptive immune responses (1). OV have been investigated as anti-cancer agents for many years and their efficacy against a variety of different tumour models has been reported. Numerous OV clinical trials have demonstrated their safety and efficacy in cancer patients.
Breast cancer (BC) is the most commonly diagnosed cancer in women in the UK and kills approximately 1000 women/month in the UK alone. Triple negative BC (TNBC) has the worst prognosis, highest death rate and lowest overall survival, of all types of BC. Therefore, novel treatments with limited toxicity and increased effectiveness are urgently required. Over the last decade, cancer immunotherapy has revolutionised cancer treatment for a range of cancers, and has the potential to do so for BC in the future. Cancer immunotherapy, and OV in particular, hold significant promise for the treatment of a range of cancers, including BC. However, BC cells have multiple mechanisms to evade immune-mediated killing, one of which is the cyclooxygenase (COX)-2 mediated production of prostaglandin E2 (PGE2), which promotes an immunosuppressive tumour microenvironment.
There is pre-clinical and clinical evidence that the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA), has anti-inflammatory and anti-cancer activity2 production by COX-2 plays a key role in anti-inflammatory properties and its anti-cancer effect. Therefore, our hypothesis is that co-administration of EPA with OV could increase therapeutic efficacy through improvement of OV-induced anti-tumour immunity. In support of this we have already shown that recombinant PGE2 inhibits oncolytic reovirus (Reolysin®)-induced immune activation, and that EPA can decrease the production of PGE2 from TNBC cells. This PhD project will establish whether EPA-downregulation of PGE2 can restore immune cell function and promote OV-induced immunotherapy.
This project will utilise sterile tissue culture techniques, for growth of cancer cell lines, fibroblasts and immune cells, flow cytometry for cell phenotyping and immunoassays, including ELISA. Ultimately this work aims to support the development of novel clinical strategies which will use EPA to potentiate OV-induced immunotherapy.
1.Parrish C, Scott GB, Migneco G, Scott KJ, Steele LP, Ilett E, et al. Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia. Leukemia 2015;29(9):1799-810.
2.Zelenay S, van der Veen AG, Bottcher JP, Snelgrove KJ, Rogers N, Acton SE, et al. Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity. Cell 2015;162(6):1257-70.
3.Cockbain AJ, Volpato M, Race AD, Munarini A, Fazio C, Belluzzi A, et al. Anticolorectal cancer activity of the omega-3 polyunsaturated fatty acid eicosapentaenoic acid. Gut 2014;63(11):1760-8.
Investigating stess hormone modulators in breast cancer treatment
Supervisors: Dr Laura C Matthews firstname.lastname@example.org, Dr Fiona Errington-Mass email@example.com, Prof Graham P Cook firstname.lastname@example.org
Breast cancer (BC) is the most common cancer, with over 1 million new cases diagnosed worldwide each year. BC is a complex disease that occurs as a result of the combined effect of environmental factors and an underlying genetic susceptibility. This means that breast cancer exists in many forms. For some types of BC, hormonal treatments have been very effective but one particular BC subtype, triple negative breast cancer (TNBC), is highly aggressive and lacks a targeted therapy. Currently the only therapeutic options for TNBC is standard radiotherapy and chemotherapy. Finding new treatment options therefore remains a major unmet clinical need.
Oncolytic viruses (OV) preferentially infect and kill cancer cells, and their clinical use has been successful for treatment of cancers such as melanoma. OV infect cancer cells and destroy them by a process called oncolysis. In doing so, new virus particles are released which infect and destroy nearby cells within the tumour. OV also stimulate systemic host anti-tumour immune responses, which help to destroy primary cancer cells and also detect and destroy circulating or metastatic disease. However, within the tumour microenvironment multiple mechanisms exist which enable cancer cells to evade immune recognition and direct destruction. One possible route is through the action of the stress hormone – known as glucocorticoid.
Glucocorticoids (Gc) are steroid hormones important for normal physiology – with major actions on energy homeostasis and immune function. The naturally occuring Gc is released into the circulation with a daily rhythm which in human peaks at the onset of waking (morning in human, evening in mice), and is also released in response to stress. Due to their potent suppressive effects on immune function, synthetic Gcs such as Dexamethasone are in wide clinical use as potent anti-inflammatory drugs. They are prescribed as part of standard therapy for various cancers - including BC - alongside standard radiotherapy and chemotherapy protocols. Gc have complex effects on cell fate and immunity, and so it is difficult to predict how Gcs, either the endogenous stress steroid cortisol or the prescribed steroid dexamethasone, might affect OV action.
To add even further complexity, the receptor that responds to Gcs - the Gc receptor, or GR - is expressed in TNBC and is under investigation as a tractable target for therapy. This is because patients with tumours that express the highest levels of GR have more chance of relapse, metastasis, and shorter survival times. This tells us that something that occurs as part of Gc signaling somehow imparts a survival benefit to TNBC cancer cells. This project will systematically characterise the effect of modulating Gc receptor expression and activation by steroid on OV function in BC. Specifically, this will be achieved by examining the effect of GR overexpression and knockdown, coupled with cortisol and dexamethasone treatment on (i) direct OV driven BC cell oncolysis, (ii) OV driven inflammation and immune activation, and (iii) bystander killing of BC by activated immune cells.
Through this, the project will generate the first direct experimental evidence to answer three key questions:
i) Whether GR expression alters the response of TNBC to OV – suggesting whether screening for GR content would be useful in stratifying patients to OV therapy. Current protocols exist to define the expression of two related receptors that bind estrogen and progesterone to guide therapy.
ii) Whether timing OV administration to the evening when cortisol is low, might increase OV anti-cancer efficacy. The concept of ‘chronotherapeutics’ which refers to timing drug administration to maximise efficacy - is particularly important as the immune system (under Gc control) is under strong time-of-day regulation.
iii) Whether prescribed steroids, such as dexamethasone, which are routinely given alongside standard cancer therapy to reduce the inflammation caused by surgery, radiotherapy and chemotherapy has a direct effect OV anti-cancer efficacy. This is important in considering their safe use as part of OV cancer therapy – and whether alternative anti-inflammatory drugs should be considered instead.
The project therefore has potential to inform new strategies for treating TNBC either by screening for GR expression, informing timing of OV protocols, and inclusion of Gc as part of standard therapy.
Training will be provided in all aspects, and so no specific previous experience is required. The project will use a range of bioinformatic, cell and molecular biology techniques to achieve the aims outlined above. This includes training in cell culture; plasmid and siRNA transfection; viral infection; cell fate assays; ELISA; PCR; flow cytometry; immunoblotting and immunofluorescent microscopy. These broad skills provide a solid basis to pursue a career in most biology fields but the appointed student will develop background knowledge and specific expertise that is particularly relevant to developing a research career in the fields of cancer biology and/or immunology.
1. Matthews LC, Berry AA, Morgan DJ, Poolman TM, Bauer K, Kramer F, Spiller DG, Richardson RV, Chapman KE, Farrow SN, Norman MR, Williamson AJK, Whetton AD, Taylor SS, Tuckermann JP, White MRH, Ray DW. (2015) Glucocorticoid receptor regulates chromosome segregation and is associated with malignancy. PNAS. 112, 5479-5484.
2. Gibbs J, Ince L, Matthews L, Mei J, Bell T, Yang N, Saer B, Begley N, Poolman T, Pariollaud M, Farrow S, DeMayo F, Hussell T, Worthen GS, Ray D, Loudon A. (2014) An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action. Nature Medicine 20, 919-926.
3. Samson A, Scott KJ, Taggart D, West EJ, Wilson E, Nuovo GJ, Thomson S, Corns R, Mathew RK, Fuller MJ, Kottke TJ, Thompson JM, Ilett EJ, Cockle JV, Van Hille P, Sivakumar G, Polson ES, Turnbull SJ, Appleton ES, Migneco G, Rose AS, Coffey MC, Beirne DA, Collinson FJ, Ralph C, Anthoney DA, Twelves CJ, Furness AJ, Quezada SA, Wurdak H, Errington-Mais F, Pandha H, Harrington KJ, Selby PJ, Vile RG, Griffin SD, Stead LF, Short SC, Melcher AA. 2018. Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade. Science Translational Medicine 10, (422).
Sun exposure as a novel risk factor for mosquito-borne disease
Supervisors: Dr Clive McKimmie email@example.com, Dr Steve Griffin & Dr Kave Shams
Mosquito-borne disease is now receiving unprecedented attention due to major outbreaks in new geographic areas including Brazil, Mexico, India, China, the Middle East and SE Asia. A changing climate and globalization is increasing their range, as witnessed by the spread of the Zika and dengue viruses. These viruses can cause highly disabling and sometimes lethal disease. In the absence of effective medications there now exists an urgent need to better understand these infections. Furthermore, the clinical course of these diseases is highly unpredictable, which makes the management of these patients difficult.
Infected mosquitoes transmit virus to the human host as they probe the skin for blood. As such, infection of the skin represents a key stage of infection that is a common aspect of all mosquito-borne virus infections. We showed, in a widely-reported paper, that host inflammatory responses to mosquito bites inadvertently enhances virus infection. Fascinatingly, we have now data that shows exposure of the skin to ultraviolet (UV) light from the sun modulates the skin response to virus infection at mosquito bites. As such, exposure to sun may modulate host susceptibility to infection by mosquito-borne viruses.
This ambitious project will work out how UV has this important modulating effect on infection. UV may help the virus infect cells directly or could also modulates immune responses to the virus or the mosquito bite.
Potentially, sun exposure could be a useful risk factor that helps clinicians to treat patients and additionally can inform public health campaigns that aim to modulate sun exposure.
This project will benefit from using the latest biomedical technologies and a unique collaborative environment that brings to together clinicians and scientists. This includes using systems biology-based methodologies to discover new personalized medicine approaches and therapies for treating these infections. As such, this project will provide the perfect opportunity to learn new skills, Systems biology, RNA-seq, in vivo models, quantitative PCR, 12-colour flow cytometry, cell sorting, tissue culture of human skin explants gain expertise from leading specialists and publish papers in world leading journals.
For more info see our website: http://www.virus-host-interactions.org
1. Mosquito Biting Modulates Skin Response to Virus Infection (2017) Marieke Pingen, Michael Schmid, Eva Harris, and Clive S McKimmie. Trends in Parasitology, Aug;33(8):645-657. https://www.ncbi.nlm.nih.gov/pubmed/28495485
2. Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection (2016) Pingen M, Bryden SR, Pondeville E, Schnettler E, Kohl A, Merits A, Fazakerley JK, Graham GJ, McKimmie CS. Immunity. 44, 1455–1469. http://www.cell.com/immunity/abstract/S1074-7613(16)30205-9. This paper received considerable media coverage, with articles in e.g. TIME, Nature and Scientific American; https://www.altmetric.com/details/8951929#score
Manipulating T cell metabolism to improve anti-tumour immunity
Supervisors: Dr Robert Salmond firstname.lastname@example.org
& Dr Mihaela Lorger
The induction of immune responses to tumours can provide long-lasting protection from cancer. In this regard, T cells can suppress tumour growth by directly killing cancer cells and by producing inflammatory cytokines. Furthermore, advances in immunotherapy have shown the adoptive cell transfer (ACT) of tumour-reactive T cells to be a successful approach to the treatment of cancer. However, in many individuals the T cell response to cancer is ineffective. It has become apparent that the processes of T cell activation and differentiation are linked to the regulation of basic metabolic pathways. These pathways provide energy required for growth, proliferation and effector functions. Dysregulation of cellular metabolism has been linked to the failure of anti-tumour T cell responses. Therefore, a greater understanding of the key pathways and regulators of T cell metabolism has the potential to define new therapeutic targets and approaches to manipulate T cell responses in the clinic. The primary supervisor’s research team has determined that a mitochondrial metabolic enzyme, PEPCK2, plays an important role in the differentiation and effector function of inflammatory CD8+ and CD4+ T cells.
This project will build upon initial findings to:
1. Determine the impact of pharmacological and genetic inhibition of PEPCK2, and the related PEPCK1, on mouse T cell activation, differentiation and effector function
2. Investigate the role of PEPCKs in T cell metabolism
3. Determine how manipulation of PEPCK2 expression might be harnessed to improve the efficacy of anti-tumour T cell responses using in vivo mouse models
1. The tyrosine phosphatase PTPN22 discriminates weak self peptides from strong agonist TCR signals. Salmond RJ, Brownlie RJ, Morrison VL, Zamoyska R. Nat. Immunol. 2014, 15:875-883
2. Resistance to TGFb suppression and improved anti-tumor responses in CD8+ T cells lacking PTPN22. Brownlie RJ, Garcia C, Ravasz M, Zehn D, Salmond RJ, Zamoyska R. Nat. Commun. 2017, 7:1343
3. mTOR regulation of glycolytic metabolism in T cells. Salmond RJ. Front Cell Dev Biol. 2018, 6:122.
Exploring the mitotic functions of ASPM in human brain size regulation
Supervisors: Dr J Bond J.Bond@leeds.ac.uk, Dr EE Morrison & Prof M Peckham
The increase in relative brain size is one of the most striking events in human evolution. To determine how human brain size is normally regulated we have investigated the cause of autosomal recessive primary microcephaly (MCPH), a congenital disorder of reduced brain size and associated mental retardation. MCPH is a disorder of reduced neural progenitor cell and neuronal cell number in the developing brain.
Homozygous mutations in the Abnormal Spindle-Microtubule Assembly gene (ASPM – previously named Abnormal Spindle -like Microcephaly associated gene) are the most common cause of MCPH (Bond et al., 2003, Am J Human Genet. 73:1170-1177). ASPM is a 410kDa microtubule associated protein which localises to the spindle poles during mitosis and to the midbody during cytokinesis (Bond et al., 2002, Nat Genet. 32:316-20; Higgins et al., 2010 BMC Cell Biol. 11:85-101). Analysis of patient cells containing ASPM mutations and cell lines in which ASPM has been reduced using siRNA have identified a number of mitotic functions for ASPM. Notably, interactions involving the C-terminus of ASPM are involved in the final stages of cytokinesis. We wish to further investigate the protein pathways involved in this.
Using cell-based siRNA library screening, Yeast-two hybrid and other interaction studies, we have identified a number of ASPM interacting proteins that we hypothesize to be involved in spindle orientation, cytokinesis, cytoskeletal organisation, structural changes in ASPM and the regulation of ASPM activity. This PhD project will validate and further study a number of these protein interactions.
The project will utilise a range of biochemical, molecular biological and cell biological techniques. Gain or loss-of-function cellular phenotypes will be assessed by over-expression and siRNA-mediated knockdown of gene expression in cultured cells, alongside analysis in a unique resource of MCPH patient primary cells. Live cell imaging, confocal microscopy, super resolution microscopy and high throughput imaging will complement these approaches.The programme of work will provide a motivated and enthusiastic post-graduate student with an excellent foundation in research training in a range of modern techniques including PCR, cloning, co-immunprecipitation, GST fusion pull down assays, siRNA gene knockdown, cell culture, RNA extraction, Real time PCR, immunofluorescence staining, Western blotting and cellular imaging of fixed and live cells.
1. Bond et al., 2003, Protein-truncating mutations in ASPM cause variable reduction in brain size. Am J Human Genet. 73:1170-1177
2. Bond et al., ASPM is a major determinant of cerebral cortical size. 2002, Nat Genet. 32:316-20
3. Higgins et al., Human ASPM participants in spindle organisation, spindle orientation and cytokinesis. 2010 BMC Cell Biol. 11:85-101