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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Investigating Immune Pathways in Cardiovascular Diseases

Supervisor: Dr Pasquale Maffia

Project outline:Immune responses play key roles in cardiovascular diseases (CVD) such as atherosclerosis and hypertension. By using a broad range of vascular, immunological and omics techniques we aim to study the net contribution of specific immune pathways to CVD in humans and experimental models.

Techniques to be used: The project will provide training in both vascular biology and immunology, including flow cytometry, microscopy and single-cell omics.

References

1. Maffia P, Mauro C, Case A, Kemper C. Canonical and non-canonical roles of complement in atherosclerosis. Nat Rev Cardiol. 2024 Apr 10. doi: 10.1038/s41569-024-01016-y.
2. Guzik TJ, Nosalski R, Maffia P, Drummond GR. Immune and inflammatory mechanisms in hypertension. Nat Rev Cardiol. 2024;21(6):396-416.
3. MacRitchie N, Grassia G, Noonan J, Cole JE, Hughes CE, Schroeder J, Benson RA, Cochain C, Zernecke A, Guzik TJ, Garside P, Monaco C, Maffia P. The aorta can act as a site of naïve CD4+ T-cell priming. Cardiovasc Res. 2020;116(2):306-316.
4. Hu D, Mohanta SK, Yin C, Peng L, Ma Z, Srikakulapu P, Grassia G, MacRitchie N, Dever G, Gordon P, Burton FL, Ialenti A, Sabir SR, McInnes IB, Brewer JM, Garside P, Weber C, Lehmann T, Teupser D, Habenicht L, Beer M, Grabner R, Maffia P, Weih F, Habenicht AJ. Artery Tertiary Lymphoid Organs Control Aorta Immunity and Protect against Atherosclerosis via Vascular Smooth Muscle Cell Lymphotoxin Beta Receptors. Immunity. 2015;42:1100-15.

School of Infection and Immunity, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8TA

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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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T cell/APC interactions and immunological decisions

Supervisors: Prof James Brewer, Prof Paul Garside

Project outline: It is becoming clear that the duration, frequency, and intensity of T cell/APC interactions, determines the induction of immunological tolerance versus priming. However, the detailed molecular mechanisms regulating cellular interactions in vivo remain unclear. We contend that spatiotemporal context has a critical influence on T/APC interactions and consequently the induction, maintenance and/or control of immune responses. For example, we have recently shown that the duration and magnitude antigen presentation and the subsequent T cell/APC interaction can influence differentiation of T cells to the Tfh phenotype responsible for driving B cell antibody production. Consequently, cellular and molecular interactions must be carefully choreographed in space and time to provide normal immune function giving protection against infection while avoiding autoimmunity. On the other hand, dysregulated spatiotemporal expression of molecules involved in T cell/APC interactions may result in pathology.

Summary aim: 

1. What are the molecular mechanisms controlling T/APC interactions during priming and tolerance in vivo?
2. How do these pathways impact on the duration, frequency and intensity of T cell/APC interactions in Lymph Nodes (LN)

Techniques to be used: High content (INCELL) imaging, Live in vitro microscope, Intravital multiphoton microscopy

References

1. Zinselmeyer, B. H. et al. In situ characterization of CD4+ T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance. J. Exp. Med. 201, 1815–23 (2005).
2. Millington, O. R. et al. Malaria impairs T cell clustering and immune priming despite normal signal 1 from dendritic cells. PLoS pathogens 3, 1380–7 (2007).
3. Celli, S., Lemaître, F. & Bousso, P. Real-time manipulation of T cell-dendritic cell interactions in vivo reveals the importance of prolonged contacts for CD4+ T cell activation. Immunity 27, 625–34 (2007)

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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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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

The immune system provides vital protection against infection, and can be manipulated by vaccination to provide life-long resistance to pathogens. However, immune and inflammatory responses also make a major contribution to a spectrum of human pathologies, from chronic inflammatory disease, allergy and autoimmunity, neuroinflammatory disorders and brain immune interactions, to heart disease and cancer. 

Research in the Centre for Immunobiology within the School of Infection and Immunity is focused on generating a molecular and cellular understanding of the immune system in health and disease, and applying this knowledge to the development of novel therapeutics. This is built on close interactions between an excellent cohort of scientists and clinicians within the Centre, and on the networks of collaborators they have established with researchers in the rest of the School, elsewhere in the university, and further afield.

Our staff and students benefit from access to state-of-the-art laboratory facilities in the Sir Graeme Davis building at the heart of the university and in clinical units in hospitals across Glasgow. We have expertise in a broad range of techniques, including molecular biology, ‘Omics, cell biology, multiparameter flow cytometry, intravital imaging, and in vivo models of disease, and these approaches allow us to explore the immune system at the molecular, cellular and whole organism level.

The PhD programme in immunobiology is based on individual research projects covering an exciting range of topics, with specific areas of interest including (in alphabetical order):

• atherosclerosis
• bioinformatics
• cancer and leukaemia
• chemokines and cell migration
• cytokine biology
• dendritic cell biology
• imaging the immune response
• infectious disease
• intestinal immunity
• intracellular signalling and transcriptional regulation
• lymphocyte biology
• neuroimmunology, including repair strategies forbrain repair following immunologically mediated injury (Multiple Sclerosis, Guillain-Barré syndrome)  and spinal cord injury using glial/stem cell transplantation and antibody profiling
• osteoimmunology
• rheumatology
• tissue injury and repair; focus on regenerative medicine

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

MD (Doctor of Medicine)

• Duration: 2 years full-time; 4 years part-time (for medically-qualified graduates only)