Centre for Biomedical Science Research PhD Scholarship Scheme

Director of Studies:
Professor Jim Boyne

Co-supervisor:
Dr Tara Sabir

Project summary:
RNA-binding proteins (RBPs) are emerging as key regulators in cancer biology. Our lab has identified a subset of circular RNAs [circRNAs] regulated by the RBP SFPQ in triple-negative breast cancer [TNBC], a clinically aggressive subtype with limited treatment options. While circRNA biogenesis remains poorly understood, recent evidence implicates SFPQ in their production and function. This PhD project will explore the molecular mechanisms underpinning SFPQ-circRNA interactions using a multidisciplinary approach that includes site-specific SFPQ mutants, RNA-binding assays, and single-molecule fluorescence microscopy.

This project offers extensive training in molecular biology, RNA biochemistry, and cutting-edge single-molecule fluorescence techniques, including smFRET and smTIRF microscopy. There will also be an opportunity to engage with bioinformatics pipelines via our collaborators. Together, these methods will be used to uncover the binding dynamics, structural changes, and regulatory mechanisms by which SFPQ interacts with target circRNAs in TNBC. This cutting-edge project will advance our understanding of how SFPQ regulates circRNA biogenesis and/or function, and by extension provide general insights into post-transcriptional gene regulation. Longer term, the findings could inform therapeutic strategies targeting disease-relevant RBP-circRNA interactions in cancer.

Contact details:
For an informal discussion please contact Professor Jim Boyne: j.boyne@leedsbeckett.ac.uk

Director of Studies:
Dr Sareen Galbraith

Project summary:
Alphaviruses are an emerging public health threat due to increasing mosquito vector distribution. A recent Eastern equine encephalitis outbreak in USA caused 35% mortality and almost half of survivors experienced long-term neurological complications. Chikungunya causes regular epidemics of arthritis and arthralgia across Asia, Africa, the Americas and Europe. Outbreaks often have high morbidity with severe joint pain persisting for months or years. Currently, there are no available therapies. Autophagy is an autonomous, tightly regulated, cellular process where damaged proteins and organelles are delivered to lysosomes for degradation in double membrane vesicles, called autophagosomes.

This proposal uses the well-established model alphavirus, Semliki Forest virus (SFV), to study the role of autophagy in virus infection and develop novel therapeutic strategies. SFV modulates autophagic processes in infected cells. We have shown that virus replication suppresses autophagy during early stages of infection to promote growth; yet this block is removed later in infection resulting in autophagosome accumulation. The autophagy pathway is regulated by mTOR and Beclin-1 protein complexes.

We will further investigate this autophagy modulation by examining the interaction of viral proteins with mTOR, Beclin-1 and cellular factors using FACS analysis and qPCR. Preliminary experiments have shown that autophagy inducing drug treatment early in infection significantly reduced virus growth augmenting the natural autophagy suppression. Development of autophagy-based therapeutics is rapidly expanding. New drugs, such as small molecule inhibitors (cediranib, magnolol), and repurposed drugs, such as pazopanib (cancer) and hamartin (ischemia-reperfusion injury) will be used to treat virus infected neuronal cells. Infectious virus production will be measured using plaque assay and Western blotting and FACS analysis of LC3 will be used to evaluate autophagic flux.

The student will gain multidisciplinary training in cell biology, virology and molecular biology, providing employment options in the NHS, industry, government and academia post qualification.  

Contact details:
For an informal discussion please contact Dr Sareen Galbraith: s.e.galbraith@leedsbeckett.ac.uk

Director of Studies:
Dr Ian Hurley

Project summary:
Justification for study - an important contributor to the incidence of fraud in the food industry is the adulteration of foods. The rights of consumers and honest food producers with regard to fraudulent adulteration are protected by law. This has become particularly apparent with the recent reports of horse and pork meat being found in beef products. Halal and Kosher meat products should be free of adulteration (or contamination) with pork. At present, there is no validated and widely adopted system in place to verify the composition of meat products and provide the assurance that they are strictly free from pork.

Aim of project - the project concerns the development of novel biomolecular assays for the sensitive and selective detection of the species of origin of meat products. A bank of assays will initially be developed which will utilise species-specific antibodies (ELISA) to porcine antigens, and primers (PCR-based methods) to porcine DNA. This data will then be used to validate novel aptamer-based approaches (ELONA) to facilitate the specific detection of the target species, an approach which confers some advantages over traditional detection methods. This aims to culminate in the production of in-house commercial lateral-flow kits for routine on-site monitoring. 

Objectives:

• ELISA - a direct sandwich anti-porcine IgG ELISA for the rapid detection of pork meat Specificity and sensitivity of the assays will be determined.
• PCR - porcine-specific primers will be used in qPCR as a confirmatory assay for food adulteration.
• ELONA - SELEX approaches will be used to identify aptamers that can used in novel ELONA strategies for food adulteration studies.

Validation - each of these assays will undergo validation. These will be performed to ascertain the robustness of the assay and gauge the suitability of the assay for the routine screening of Halal and Kosher products for the presence of pork.  

References:

1. Eldahshoury, M. K. and I. P. Hurley (2023). "Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk." Journal of Dairy Science 106(9): 5908-5915.

Contact details:
For an informal discussion, please contact Dr Ian Hurley: i.p.hurley@leedsbeckett.ac.uk

Director of Studies:
Professor Gary Jones 

Project summary:
Gliotoxin (GT) is a sulphur-containing mycotoxin that inhibits fungal and bacterial growth. It can exist in a reduced form called dithiol gliotoxin (DTG). Recently we demonstrated that DTG was capable of binding zinc and that the toxicity of GT/DTG to cells was dependent on the availability of zinc in the environment [Saleh et al. 2018]. This led to the proposal of utilising GT/DTG and its zinc (and other metal ion) binding ability as a pathfinder molecule to identify new targets and cellular pathways for overcoming antimicrobial resistance [Downes et al. 2023a]. However, the complex relationship between GT/DTG and its ability to bind metal ions, and the biological implications of this, are not yet fully understood.

The bacterial pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter. baumannii, Pseudomonas aeruginosa and Enterobacter spp. comprise the ESKAPE group of bacteria and have been designated “highest priority” by the World Health Organisation for the research and development of new antibiotics. A baumannii is one of the most challenging species to treat due to its overwhelming arsenal of strategies to overcome antibiotic therapy. In published and preliminary work, we have discovered that GT/DTG directly inhibits A. baumannii growth in a metal-dependent manner [Downes et al. 2023b]. Through a series of inhibitory and proteomics studies we had identified proteins and cellular pathways that may be suitable as novel antimicrobial targets.

The aims of this project are twofold:

• To use the model organism Saccharomyces cerevisiae [yeast] as an experimental tool to systematically assess the in vivo biological relevant relationship of GT/DTG and interaction with metal ions. This will be achieved through a series of targeted experiments exposing yeast to varying concentration of GT/DTG in the presence and absence of specific metal ions, and analysing how cells respond using genetics, biochemistry, molecular biology, proteomics and genomics.
• Identifying potential new antibacterial targets to counteract AMR in the ESKAPE pathogen baumannii. Initial work will involve the identification of possible targets from analysis of unpublished proteomics data, followed by targeted experiments with A. baumannii based on the biological and cellular roles of the target genes/proteins.

This project is part of an ongoing collaboration between Professor Jones and colleagues at Maynooth University in Ireland. Some of the work will require a prolonged stay (minimum 3 months) in the laboratory of Professor Sean Doyle, so only apply for this project if you are prepared to carry out this work placement.

References

1. Saleh A. A, G. W. Jones, F. C. Tinley, S. F. Delaney, S. Alabbadi, K. Fenlon, S. Doyle and R. O. Owens (2018) Systems impact of zinc chelation by the epipolythiodioxopiperazine dithiol gliotoxin in Aspergillus fumigatus: a new direction in natural product functionality. Metallomics. doi: 10.1039/c8mt00052b.
2. Downes, S.G, S. Doyle, G. W. Jones and R. Owens (2023a) Gliotoxin and related metabolites as zinc chelators: implications and exploitation to overcome antimicrobial resistance. Essays in Biochemistry Sep 13;67(5):769-780. doi: 10.1042/EBC20220222. (REVIEW ARTICLE)
3. Downes S. G, R. A. Owens, K. Walshe, D. A. Fitzpatrick, A. Dorey, G. W Jones and S. Doyle (2023b) Gliotoxin-mediated bacterial growth inhibition is caused by specific metal ion Scientific Reports, Sep 27;13(1):16156. doi: 10.1038/s41598-023-43300-w.

Contact details:
For an informal discussion please contact Professor Gary Jones: gary.jones@leedsbeckett.ac.uk

Director of Studies:
Dr Tara Sabir

Co-supervisor:
Dr Donna Johnson

Project summary:
smTIRF (single molecule Total Internal Reflection fluorescence) is a well-established tool able to offer a novel approach to understanding the intricacies of small molecules interacting with each other at a high molecular resolution. The approach involves chemically incorporated fluorescent dyes and surface immobilisation via a constructed microfluidic device. Thus providing us with unique insights into molecular behaviour molecule by molecule. smFRET (single molecule Förster Resonance Energy Transfer) is adapted to follow the structural changes and dynamics of interactions (1).

In particular we will be looking to use this experimental approach to further understand the structural formation, dispersion and virulence of biofilms. In the natural world biofilm formation has many benefits for the organism. However biofilm formation on medical devices, for example catheters and/or implants, used post operation are a major cause for concern and harm to patient recovery. Although improvements regarding how to tackle this issue through a clearer understanding of biofilm formation is well documented there are still unanswered questions which would benefit from the high resolution that smTIRF has to offer (2).

This project will be part instrument build and part biological investigation and as such requires a good understanding of optics, microscopy, fluorescence spectroscopy, biochemistry and software writing. Additionally a strong willingness to work collaboratively between institutions and disciplines will be expected. Candidates should have a first degree in Physics, Biophysics or a related Biosciences degree with a strong interest in Biochemistry.

References:

1. Colson L, Kwon Y, Nam S, Bhandari A, Maya NM, Lu Y & Cho Y. Trends in Single-Molecule Total Internal Reflection Fluorescence Imaging and Their Biological Applications with Lab-on-a-Chip Technology. Sensors (2023): vol 23 (18), pg 7691.
2. Percival SL, Suleman L, Vuotto C & Donelli G. Healthcare-associated infections, medical devices and biofilms: risk, tolerance and control. Journal of Medical Microbiology (2015), vol 64(4) pg 323-334.

Contact details:
For an informal discussion please contact Dr Tara Sabir: t.sabir@leedsbeckett.ac.uk