Research projects

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A new role for the TPL-2 kinase in the nucleus

Supervisor: Ruaidhrí Carmody

Project description:

The TPL-2 kinase is a key activation of mitogen activated protein kinases (MAPK) in innate immune cells and is required for the inducible production of inflammatory cytokines during infection. Mutations in TPL-2 can lead to cancer through it’s ability to promote cell proliferation and survival. The activity of TPL-2 is normally tightly controlled and active TPL-2 is rapidly degraded to limit the activation of downstream pathways. We previously demonstrated that TPL-2 is a nuclear-cytoplasmic shuttling protein that undergoes ubiquitination and proteasomal degradation in the nucleus. However, whether TPL-2 plays any functional role in the nucleus is currently unknown. Published studies and preliminary data generated within the Carmody lab indicates that TPL-2 may interact with and phosphorylate nuclear proteins, including transcription factors. This project will investigate the nuclear function of TPL-2 in regulating inflammatory responses and how it might contribute to the oncogenic effects of TPL-2 mutation.
The results of this project will be highly relevant to understanding the regulation of inflammation by TPL-2 and the consequences of its mutation in cancers.

Summary aim: This project will investigate the nuclear function of TPL-2 kinase.

Techniques: Cell culture, protein-protein interaction studies, CRISPR/Cas9 gene editing, transcriptomic analysis by QPCR and RNA-seq.

References:

1. P. E. Collins, D. Somma, D. Kerrigan, F. Herrington, K. Keeshan, R. J. B. Nibbs, R. J. Carmody, The IκB-protein BCL-3 controls Toll-like receptor-induced MAPK activity by promoting TPL-2 degradation in the nucleus. Proc. Natl. Acad. Sci. 116, 25828–25838 (2019).

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Decoding temporal windows for mitochondrial intervention across species using data-driven approaches 

Supervisor: Alberto Sanz

Project description: 

Mutations in genes encoding mitochondrial OXPHOS components often cause devastating diseases. Paradoxically, lifespan can be extended in worms, flies, and mice by partially impairing mitochondrial function. Why this occurs remains poorly understood. The Sanz laboratory has revealed that the timing of mitochondrial dysfunction is critical—similar interventions produce opposing outcomes depending on when they are applied. This project aims to systematically identify conserved patterns that explain this temporal dependency.

We will analyse RNAseq and proteomics datasets from Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, and Homo sapiens, comparing short- and long-lived species. We will assess mRNA and protein expression across tissues and developmental stages, quantifying correlations between transcript and protein levels. Using machine learning, we will identify conserved expression profiles that predict lifespan outcomes.

Guided by these insights, we will use state-of-the-art genome editing in Drosophila to modulate mitochondrial function with spatiotemporal precision, targeting specific tissues and developmental windows to generate new long-lived flies.

By uncovering the timing and location rules that govern mitochondrial influence on longevity, this work will establish a foundation for future interventions to extend human healthspan.

Techniques:

• Data mining
• Transcriptomics
• Proteomics
• Statistical Analysis
• Machine Learning
• Genetics
• Molecular Biology

References:

1. Developmental mitochondrial complex I activity determines lifespan. Stefanatos R, Robertson F, Castejon-Vega B, Yu Y, Uribe AH, Myers K, Kataura T, Korolchuk VI, Maddocks ODK, Martins LM, Sanz A. EMBO Rep. 2025 Apr;26(8):1957-1983

2. NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype. Muela-Zarzuela I, Suarez-Rivero JM, Gallardo-Orihuela A, Wang C, Izawa K, de Gregorio-Procopio M, Couillin I, Ryffel B, Kitaura J, Sanz A, von Zglinicki T, Mbalaviele G, Cordero MD. Inflamm Res. 2024 Aug;73(8):1253-1266.

3. Autophagy promotes cell survival by maintaining NAD levels.
   Kataura T, Sedlackova L, Otten EG, Kumari R, Shapira D, Scialo F, Stefanatos R, Ishikawa KI, Kelly G, Seranova E, Sun C, Maetzel D, Kenneth N, Trushin S, Zhang T, Trushina E, Bascom CC, Tasseff R, Isfort RJ, Oblong JE, Miwa S, Lazarou M, Jaenisch R, Imoto M, Saiki S, Papamichos-Chronakis M, Manjithaya R, Maddocks ODK, Sanz A, Sarkar S, Korolchuk VI.
   Dev Cell. 2022 Nov 21;57(22):2584-2598.e11.

4. Mitochondrial ROS signalling requires uninterrupted electron flow and is lost during ageing in flies. Graham C, Stefanatos R, Yek AEH, Spriggs RV, Loh SHY, Uribe AH, Zhang T, Martins LM, Maddocks ODK, Scialo F, Sanz A. Geroscience. 2022 Aug;44(4):1961-1974.

5. Mitochondrial complex I derived ROS regulate stress adaptation in Drosophila melanogaster. Scialò F, Sriram A, Stefanatos R, Spriggs RV, Loh SHY, Martins LM, Sanz A.Redox Biol. 2020 May;32:101450. 

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Exploring the roles of methionine sulfoxide reductases in mammalian cells 

Supervisor: Brian Smith

Project description:

Methionine oxidation is an enigmatic, stereo specific posttranslational modification of proteins that is traditionally thought to be a result of oxidative damage. Cells express enzymes that act as methionine sulfoxide reductases (Msr) with two distinct families, MsrAs and MsrBs acting on the S and R epimers, respectively. Intriguingly, the mammalian endoplasmic reticulum, an oxidising compartment of the cell, is only known to contain one Msr, MsrB3, leaving open the question as to whether methione-S-sulfoxide can be repaired in this location and indeed whether MsrB3 acts as a reductase or an oxidase. Defects in MsrB3 expression or function are linked to health conditions including deafness indicating that it is worthy of further study. In this project, you will explore MsrB3 function at the structural and cellular level exploiting new chemical biological tools in collaboration with Prof Hartley (School of Chemistry).

Techniques used:

• Biomolecular NMR spectroscopy,
• Protein X-ray crystallography,
• Biochemical assays,
• Chemical biology tool compounds,
• Cell biology

References:

1. Cao Z, Mitchell L, Hsia O, Scarpa M, Caldwell ST, Alfred AD, Gennaris A, Collet JF, Hartley RC, Bulleid NJ. Methionine sulfoxide reductase B3 requires resolving cysteine residues for full activity and can act as a stereospecific methionine oxidase. Biochem J. 2018 Feb 28;475(4):827-838. doi: 10.1042/BCJ20170929.

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Phosphorylation of the NF-κB transcription factor and its role in regulating the inflammatory response

Supervisor: Ruaidhrí Carmody

Project description: 

The NF-kB family of transcription factors is a key mediator of inflammation and a critical determinant of human health and disease. NF-κB mediated transcription of genes that promote cell survival and proliferation, and the emerging importance of inflammation in diseases such as cancer, atherosclerosis and neurodegeneration, establishes NF-κB as a pathological factor of ever increasing importance. Unfortunately, inhibitors of NF-κB activation have failed to make a clinical impact due to severe unwanted side-effects, likely resulting from homeostatic roles of NF-κB. Thus, despite the tremendous therapeutic promise of modulating it’s activity, NF-κB remains an important target of untapped potential.

This project builds on previous work from our laboratory and from others that revealed the control of NF-κB-mediated transcription by site specific phosphorylation. Preliminary data show that phosphorylation of NF-κB at specific sites regulates interactions with other transcription factors suggesting a mechanism for the gene selective control of transcription by NF-κB phosphorylation. This proposal aims to define the role of NF-κB phosphorylation in controlling transcriptional responses and the functional consequences of disrupting it using proteomic, transcriptomic, cell and molecular biology approaches. The data generated will be relevant to our understanding of human diseases where NF-κB has been demonstrated to be important, such as inflammatory disease and cancer, and will be important in directing future therapeutic strategies aimed at inhibiting inflammation.

Summary aim: This project will investigate the phosphorylation of the NF-κB transcription factor.

Techniques: Cell culture, protein-protein interaction studies, proteomics, CRISPR/Cas9 gene editing, transcriptomic analysis by QPCR and RNA-seq.

References:

1. F. Christian, E. Smith, R. Carmody, The Regulation of NF-κB Subunits by Phosphorylation. Cells 5, 12 (2016).
2. E. L. Smith, D. Somma, D. Kerrigan, Z. McIntyre, J. J. Cole, K. L. Liang, P. A. Kiely, K. Keeshan, R. J. Carmody, The regulation of sequence specific NF-κB DNA binding and transcription by IKKβ phosphorylation of NF-κB p50 at serine 80. Nucleic Acids Res. 47, 11151–11163 (2019).

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Structural systems biology of Staufen1:RNA transactions 

Supervisor: Laura Spagnolo 

Outline: Since its discovery, Staufen1 has been studied for its involvement in a diverse set of aspects of RNA metabolism, ranging from RNA localisation to decay. Given its pivotal role in cellular RNA metabolism, several studies have explored the mechanistic impact of Staufen1 in a wide variety of cell functions ranging from cell growth to cell death, as well as in various disease states. This PhD project aims to identify the molecular mechanism governing the functions of Staufen1 protein, using our unique expertise in the structural and quantitative biology of protein:nucleic acid complexes, track record on Staufen1 structural biochemistry, and access to beyond-state-of-the-art technology.

To gain quantitative molecular knowledge on the formation of Staufen1:RNA complexes, this project will focus on the detailed individual analysis of Staufen1:RNA complexes in vitro; as well as on gathering detailed information of such assemblies combining light and electron microscopy approaches.

Techniques

• Gene cloning, Protein over-expression purification, biochemical and cellular assays
• Reconstitution of multi-protein and protein:RNA complexes
• Assay development
• Cryo-electron Microscopy
• SAXS
• FRET; FRET-FLIM
• Super-resolution microscopy

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The control of human immune responses by the IκB proteins

Supervisor: Ruaidhrí Carmody

Project description: 

The NF-κB transcription factor and the pathways leading to it’s activation are key mediators of inflammation and immunity. The critical role of NF-κB in immunity is highlighted by the large number of human immunodeficiencies involving mutations in key components of the NF-κB pathway. This project investigates a key family of NF-κB regulators, the IκB proteins, to reveal how alterations in their expression control immune cell function. The degradation of IκB proteins is an essential step in the activation of NF-κB and is required for the transcription of hundreds of genes that control cell function and survival. Our studies have revealed that in humans different immune cell types have different profiles of IκB protein expression, suggesting cell-type specific roles for individual IκB proteins that are not currently understood. This project will use CRISPR/Cas9 methodology and our expertise in NF-κB and immunology to dissect the roles of IκB proteins in controlling human immune cell responses. We expect to identify cell-specific mechanisms that control activation and illuminate potential approaches to differentially regulate cell-specific responses. The outcomes will be highly relevant to the study of inflammatory disease, as well as the development of new therapies such as vaccines and immunotherapies.

Summary aim: This project will investigate the regulation of human immune cell activation by IκB proteins.

Techniques: Cell culture, isolation of immune cells including monocytes and T cells, CRISPR/Cas9 gene editing, RNA-seq, signal transduction analysis by immunoblotting and immunofluorescence microscopy.

References:

1. F. O. Kok, H. Wang, P. Riedlova, C. S. Goodyear, R. J. Carmody, Defining the structure of the NF-ĸB pathway in human immune cells using quantitative proteomic data. Cell. Signal. 88, 110154 (2021).
2. J. P. Mitchell, R. J. Carmody, “NF-κB and the Transcriptional Control of Inflammation” in International Review of Cell and Molecular Biology (Elsevier, 2018; https://linkinghub.elsevier.com/retrieve/pii/S1937644817300825)vol. 335, pp. 41–84.

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The molecular basis of cell self-renewal and pluripotency in embryonic stem cells

Supervisor: Laura Spagnolo 

Outline: Self-renewal and pluripotency are the main characteristics of embryonic stem cells (ESCs). Nanog is the key transcription factor that controls both self-renewal and pluripotency of ESCs.

The aims of this project are to understand on a biochemical and molecular level how Nanog self-assembles and binds to DNA. This project will involve expressing and purifying Nanog-containing macromolecular complexes, developing protocols for studying their three dimensional structure, and designing biophysical characterisation experiments (in both single molecule and ensemble setups) to unravel the molecular determinants of Nanog functions.

Techniques

• Gene cloning, Protein over-expression purification, biochemical and cellular assays
• Reconstitution of multi-protein and protein:DNA complexes
• Assay development
• Cryo-electron Microscopy
• SAXS
• FRET; FRET-FLIM
• Super-resolution microscopy

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Ubiquitin signals in Parkinson’s disease

Supervisor: Helen Walden  

Outline: Ubiquitin signalling controls almost every cellular process in humans, including targeting poorly folded proteins for destruction to prevent them harming the cell. When ubiquitin signalling goes wrong, many different disease states can occur, including neurodegenerative disorders such as Parkinsons Disease (PD).

One gene associated with PD is parkin, which gives rise to an enzyme responsible for tagging many different proteins with ubiquitin. The aims of this project are to understand on a biochemical and molecular level how parkin targets a specific substrate, Miro1, and how the ubiquitin signals applied to Miro1 are edited & interpreted.

This project will involve expressing and purifying multiprotein complexes, and developing protocols for generating the ubiquitin signals, and enzymatic assays for the regulation of these signals.

Techniques

• Cloning and sequencing
• Protein expression purification, and biochemistry
• Assay development
• X-ray crystallography
• Cryo Electron Microscopy
• Analysis of high resolution structures

References

1. Gundogdu M, Tadayon R, Salzano G, Shaw GS, Walden H. A mechanistic review of parkin activation. Biochim Biophys Acta Gen Subj. 2021 Jun;1865(6):129894. doi: 10.1016/j.bbagen.2021.129894.20.PMID: 33753174
2. Kumar A, Chaugule VK, Condos TEC, Barber KR, Johnson C, Toth R, Sundaramoorthy R, Knebel A, Shaw GS, Walden H. Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity. Nat Struct Mol Biol. 2017 May;24(5):475-483. doi: 10.1038/nsmb.3400. Epub 2017 Apr 17.PMID: 28414322

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Understanding the effects of potassium ions on biomolecular & cellular structure & function 

Supervisor: Brian Smith

Project description:

Potassium is the most abundant intracellular monovalent cation, but its effects on biomolecular structure and function are only known for a limited number of cases. Techniques that can observe the direct interaction between potassium ions and biomacromolecules have so far been limited to studying high affinity binding sites. In this project you will develop novel NMR based methods to observe low affinity interactions directly and relate them to structural and functional outcomes. In parallel, you will develop new sensors for intracellular potassium concentration leveraging AI based protein design algorithms.

Techniques used are Biomolecular NMR spectroscopy and Artificial Intelligence based protein design.

References:

1. Torres Cabán, C. C., Yang, M., Lai, C., Yang, L., Subach, F. V., Smith, B. O. , Piatkevich, K. D. and Boyden, E. S. (2022) Tuning the sensitivity of genetically encoded fluorescent potassium indicators through structure-guided and genome mining strategies. ACS Sensors, 7(5), pp. 1336-1346. (doi: 10.1021/acssensors.1c02201)

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What are the signals that determine innate immune memory?

Supervisor: Ruaidhrí Carmody

Project description:

Cells of the innate immune system can adapt to and remember previous encounters with infectious or inflammatory challenges. This innate immune memory is triggered by microbial and damage associated ligands that signal through pathogen recognition receptors. These signals lead to epigenetic changes in chromatin that modify how cells respond to a subsequent challenge by changing how strongly genes are expressed or the types of genes that are expressed. Innate immune memory can lead to an enhanced or repressed response depending on the pathogen recognition receptor ligand encountered as well as the concentration of ligand present. Memory responses that enhance inflammation can protect against infection and cancer but can also promote inflammation-associated tissue damage and chronic inflammatory disease. Conversely, memory responses that repress inflammation can protect against excessive inflammation but lead to susceptibility to infection. Thus, the innate immune memory state adopted can have significant impact on health and disease.
This project will investigate the intracellular signals triggered by pathogen recognition receptors leading to distinct memory states that either enhance or repress responses to subsequent challenge. Experiments will be performed in macrophages using techniques to analyse, activate and inhibit specific signals to understand the molecular mechanisms underpinning this fundamental aspect of innate immunity. The results will be highly relevant to understanding the regulation of inflammation as well as the development of new therapeutic strategies to treat inflammatory disease.

Summary aim: This project will investigate the signals that determine innate immune memory.

Techniques: Cell culture, high throughput immunofluorescence microscopy, mRNA transfection, CRISPR/Cas9 gene editing, ELISA, transcriptomic analysis by QPCR and RNA-seq.

References:

1. P. E. Collins, R. J. Carmody, The Regulation of Endotoxin Tolerance and its Impact on Macrophage Activation. Crit. Rev. Immunol. 35, 293–323 (2015)
2. S. K. Butcher, C. E. O’Carroll, C. A. Wells, R. J. Carmody, Toll-Like Receptors Drive Specific Patterns of Tolerance and Training on Restimulation of Macrophages. Front. Immunol. 9, 933 (2018).
3. C. O’Carroll, A. Fagan, F. Shanahan, R. J. Carmody, Identification of a Unique Hybrid Macrophage-Polarization State following Recovery from Lipopolysaccharide Tolerance. J. Immunol. 192, 427–436 (2014).
4. P. E. Collins, D. Somma, D. Kerrigan, F. Herrington, K. Keeshan, R. J. B. Nibbs, R. J. Carmody, The IκB-protein BCL-3 controls Toll-like receptor-induced MAPK activity by promoting TPL-2 degradation in the nucleus. Proc. Natl. Acad. Sci. 116, 25828–25838 (2019).

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Overview

Our biochemists and molecular biologists study the “molecules of life”, the essential molecular components of all living organisms. We aim to understand how these molecules perform their functions, using a variety of modern molecular and biochemical approaches including structural analysis at the atomic level by X-ray crystallography, NMR spectrometry, and other biophysical methods. The knowledge gained by this research gives us opportunity to invent and develop novel ways of altering biological processes to our advantage, with applications in molecular medicine, biotechnology, synthetic biology, as well as industry.

PhD programmes in biochemistry and biotechnology will carry out a cutting-edge research project in an area that aligns with the expertise of one or more of our principal investigators in the fields of biochemistry and biotechnology. The subject of the project may be fundamental “blue skies” science or may be targeted at an important application. Projects may also be related to basic science and integrate with our existing research themes, while other projects are more focused on translational aspects of our research.

Some of our current research areas are:

• cell signalling mechanisms in mammals, plants and insects
• mitochondrial biogenesis and mitochondrial proteins
• mechanisms of DNA sequence rearrangements
• DNA sequences in human disease
• genetic circuits and switches for synthetic biology
• plant molecular biology
• photosynthesis, plant photobiology, circadian factors in plants
• structural determination by NMR and X-ray crystallography
• structural bioinformatics, molecular modelling
• drug receptors, molecular pharmacology
• nuclear genomic architecture
• mechanisms of intracellular trafficking
• protein folding, targeting and modification
• protein-protein and protein-DNA interactions
• cell-surface interactions

Our PhD programme provides excellent training in cutting edge technologies that will be applicable to career prospects in both academia and industry. Many of our graduates become postdoctoral research associates while others go on to take up positions within industry, either locally or overseas. We have strong academic connections with many international collaborators in universities and research institutes.

Funds are available through the College of Medical, Veterinary and Life Sciences to allow visits to international laboratories where part of your project can be carried out. This provides an excellent opportunity for networking and increasing your scientific knowledge and skill set.

Study options

PhD

• Duration: 3/4 years full-time; 5 years part-time

Individual research projects are tailored around the expertise of principal investigators.

MSc (Research)

• Duration: 1 year full-time; 2 years part-time