Research Opportunities

The Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that control diverse signalling pathways affecting gene-transcription, cellular adhesion and trafficking, autophagy and metabolism via the generation of PIP3. While some of these readouts are controlled by the evolutionarily conserved PI3K-AKT-FOXO, PI3K-AKT-mTOR axes, there is a diverse network of PI3K effectors that are less well studied, especially in lymphocytes, but which nonetheless can have profound effects on lymphocyte biology. We have recently used CRISPR/Cas9 to perform a targeted screen of PI3K effectors by generating a library that specifically targets PIP3-binding proteins. Screening for genes that affect T cell adhesion, we identified RASA3 as a key protein linking PI3K to the activation of the integrin LFA-1 and found that RASA3 is critical for T cell migration, homeostasis and responses to immunization (Johansen et al Sci Signaling 2022; Trend Immunol 2023; Front Immunol. 2021). We have now generated extended CRISPR/Cas9 libraries that target the entire PI3K-ome (including the kinases, phosphatases and all known effector proteins). Potential projects include designing and implementing new screens for downstream readouts of PI function, including autophagy, endocytosis, regulation of humoral immunity in vivo or other readouts, and/or understanding how RASA3 and Kindlin3 regulate T cell function and the signaling pathways. Interestingly, while RASA3 and Kindlin3 are both regulated by PIP3, they have opposite effects on LFA1 activity.  We will use advanced imaging technologies to determine the differential effects of PI3K signalling on these two proteins.

Antiviral restriction factors (ARF) are a critical element of cellular innate immunity, representing the first barrier to viral infection that can determine outcome. We aim to identify and characterise novel ARF and their viral antagonists, since therapeutic interruption of viral antagonism can enable restoration of endogenous antiviral activity.

We employ a number of human pathogens, in particular Human Cytomegalovirus (HCMV), Monkeypox virus (MPXV) and its vaccine, Modified Vaccinia Ankara (MVA). Our systematic proteomic analyses determine which cellular factors each pathogen targets for destruction, since we have shown these to be enriched in novel ARFs. For example, we recently developed a multiplexed proteomic technique that identified proteins degraded in the proteasome or lysosome very early during HCMV infection (Nightingale et al, Cell Host & Microbe 2018). A shortlist of 35 proteins were degraded with high confidence, and we have since shown that several are novel ARF, with characterisation of these factors forming ongoing projects. Application to MVA infection indicated further candidates, and identified novel mechanisms of vaccine action (Albarnaz et al, Nature Communications 2023); a study on MPXV will shortly be submitted. Furthermore, interactome screens can identify the viral factor(s) responsible for targeting each ARF, and indicate mechanism (Nobre et al eLife 2019).

This project will now identify and characterise critical pan-viral ARF, which can restrict diverse viruses. For the most potent, we will determine both the mechanism of restriction and the mechanism of virally mediated protein degradation. In order to prioritise the most important factors, there will also be the opportunity to use novel multiplexed proteomic screens.

This is an opportunity to work with Prof Fitzgerald and her group who are internationally renowned for their work on Barrett’s oesophagus including the development of a novel, non-endoscopic capsule sponge test, and Dr. Katki’s group leaders in developing quantitative methods for risk-based approaches to cancer screening.

The incidence of oesophageal cancer has increased rapidly in the past 30 years. Oesophageal adenocarcinoma (OAC), the most common form of oesophageal cancer in the US and UK, has <20% 5-year survival, which improves with early detection. Individuals known to have Barrett’s oesophagus, an asymptomatic precursor condition to oesophageal adenocarcinoma, undergo surveillance with the goal of treating advanced (dysplastic) Barrett’s oesophagus before it develops into cancer, thereby preventing cancer. Even if a cancer is not prevented, surveillance may detect it an earlier, asymptomatic, stage, where survival is better. However, currently the vast majority of Barrett’s oesophagus cases are undetected. The recent development of the capsule-sponge (Cytosponge) by Prof Fitzgerald’s lab has made screening for Barrett’s oesophagus more accessible, since it is cheaper than endoscopy, the prior screening method, and can be performed at a GP practice. We therefore expect an increase in the number of people with detected Barrett’s oesophagus, and therefore the number of people undergoing surveillance.  

Current surveillance guidelines depend only on characteristics of the Barrett’s oesophagus, and have not been updated to reflect the use of capsule-sponge as a surveillance tool. We will model the time to progression among patients with Barrett’s oesophagus, to inform surveillance intervals. Since Barrett’s oesophagus is asymptomatic, it can only be detected at times when the oesophagus is evaluated; special statistical methods are therefore required to model this, such as prevalence-incidence models, developed by Dr Katki’s group. The estimates will focus on absolute risk estimates, since ideally individuals would have a surveillance visit when their absolute risk exceeded a threshold, following the concept of ‘equal management of equal risk’. 

The projects will use clinical data from large cohorts of individuals undergoing endoscopy surveillance in England, Scotland and Northern Ireland. We will use both capsule-sponge and endoscopy data to inform surveillance intervals, including how surveillance intervals could vary based on the number of previous surveillance visits. 

Innate immune responses are the first line of defense against viral infection, but their inappropriate activation can cause autoinflammatory disease. The Best and Modis groups study how the host senses viruses, mounts sufficiently sensitive yet specific responses, and how this balance can be perturbed for example by disease mutations. Our work focuses largely on the roles of two key families of viral restriction factors, the TRIMs and RIG-I-like receptors (RLRs). We integrate an exceptionally broad spectrum of approaches, from in vivo work in high and maximum contain laboratories to state-of-the-art electron microscopy. This uniquely positions us to obtain a more complete understanding of virus-host interactions under physiological conditions with mechanistic insights in atomic-level detail.  

This PhD project will focus on unravelling important insights into how RNA viruses are detected and targeted by their hosts. The aims of this project will apply our full complement of approaches, including electron microscopy, biochemical and cell-based assays, and in vivo work as needed to obtain a detailed mechanistic understanding of the contributions of virus-host interactions to antiviral innate immunity and virus pathogenesis. Our long-term goal is to use contribute to the design of novel therapeutics, such as antivirals, vaccine adjuvants, or immunomodulatory therapeutics, with potential applications in the treatment of infection, autoinflammatory disorders and cancer.

Our teams at the NIAID Rocky Mountain Laboratories and University of Cambridge are strongly committed to fostering a supportive and inclusive work environment in which trainees can thrive and experience the thrill of scientific discovery.

References:
Chiramel AI, Meyerson NR, McNally KL, Broeckel RM, Montoya VR, Méndez-Solís O, Robertson SJ, Sturdevant GL, Lubick KJ, Nair V, Youseff BH, Ireland RM, Bosio CM, Kim K, Luban J, Hirsch VM, Taylor RT, Bouamr F, Sawyer SL, Best SM (2019) TRIM5α Restricts Flavivirus Replication by Targeting the Viral Protease for Proteasomal Degradation. Cell Rep 27:3269  PMC8666140

Yu Q, Herrero del Valle A, Singh R, Modis Y (2021) MDA5 autoimmune disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA. Nat Commun 12:6668  PMCID: PMC8602431

Stoll GA, Pandiloski N, Douse CH, Modis Y (2022) Structure and functional mapping of the KRAB-KAP1 repressor complex. EMBO J 41:e111179  PMC9753469

Shannon JG, Sturdevant GL, Rosenke R, Anzick SL, Forte E, Preuss C, Baker CN, Harder JM, Brunton C, Munger S, Bruno DP, Lack JB, Leung JM, Shamsaddini A, Gardina P, Sturdevant DE, Sun J, Martens C, Holland SM, Rosenthal NA, Best SM (2023) Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19. Nat Commun 14:4481  PMC10368652

The Mughal Laboratory (The Neurovascular Research Unit) studies the neurovascular coupling mechanisms involved in regulation of blood flow in the brain and clearance of metabolic by-products. Along with providing the basic understanding of these mechanisms in physiology, the research also extends to the vascular cognitive impairment and dementia (VCID) including stroke and CADASIL. By using pre-clinical models and cutting-edge imaging approaches, the Mughal laboratory provides a thorough understanding of different neurovascular mechanisms along with the contributions of different vascular compartments (arteries—capillaries— veins) with the aim to extend this knowledge from physiology to the disease models.

The research program is supported by multiple on-going projects. Students will have the option to work on any project in the lab, and to take it in new directions. 
 

Research keywords: Neurovascular, Ion channels, Calcium signaling, Blood flow in the brain
 

During vertebrate body axis elongation, populations of progenitor cells in the posterior-most tailbud region of the embryo continually make choices about which cell type they should differentiate into. These decisions must be carefully balanced against the rates of expansion of anterior structures such as the spinal cord, notochord and somites so that a well-proportioned body axis is generated. We hypothesise that anterior tissue expansion generates force production in the tailbud that is sensed by the progenitors to regulate their rates of differentiation and movement. We have preliminary data showing the activation of key mechano-transduction pathways within the tailbud, and mutant zebrafish lines where regulators of this pathway are disrupted. The project will characterise these mutants using light-sheet imaging to test the hypothesis that cells actively respond to changes in their mechanical environment to time their addition to the elongating body axis.

We are also interested in developing projects using chick embryos as a model where we can ask how the timing of progenitor contribution is alters as the progenitor domain matures during development. In parallel we make us of embryonic organoids from mouse embryonic stem cells (gastruloids) to experimentally manipulate the mechanical and metabolic environment of cells to test how these factors modulate the timing of mesoderm cell migration and differentiation.

Quantitative imaging and pooled CRISPRi screening of single cells to understand transcription factor signaling dynamics. NF-κB is a master regulator of inflammation, immunity, and cell stress responses. The temporal dynamics of NF-κB signaling capture pathogen-specific information and govern the corresponding gene expression patterns. Recent studies have revealed that distinct NF-κB signaling profiles can lead to specific epigenetic modifications within potential enhancer regions of the genome, potentially establishing epigenetic memory for subsequent infections.

In this project, our goal is to investigate the impact of epigenetic perturbations on NF-κB signaling dynamics. We will utilize CRISPRi and a pool-genetic approach to systematically disrupt all potential enhancer regions around NF-κB-regulated genes. Additionally, we will conduct live-cell microscopy to quantitatively measure the resulting changes in NF-κB signaling dynamics. The insights gained from this study will illuminate the functions of the enhancer regions of NF-κB-regulated genes and will provide information on how tissue-specific NF-κB signaling is shaped by the epigenome, through the formation of epigenetic memories. The student will learn various interdisciplinary methods involving cell culture, quantitative microscopy, fluorescent reporter assays, automated single-cell analysis, molecular biology, and imaging data analysis.

Mechanisms of perception and cognition 
Section on Perception, Cognition, and Action, Laboratory of Sensorimotor Research (NEI/NIMH)

Students would have the option to work on any project in the lab, and to take it in new directions. Current projects in the lab aim to understand the normal brain processes by which physical signals that impinge on the sensory apparatus (eyes, ears) are transformed into perceptions, thoughts, and actions. Work in the lab has been especially invested in developing color as a model system. The advantage of color is that its physical basis (wavelength) is well characterized, yet these chromatic signals support not only low-level visual abilities such as color matching but also high-level cognitive processes such as categorization, memory, social cognition, and emotion. This variety of phenomena provides a rich opportunity for investigating the full scope of perceptual and cognitive computations that make human vision such an important source of information about the world. The lab uses many research techniques, including psychophysics and non-invasive brain imaging (MRI, MEG) in humans, along with fMRI-guided microelectrode recording, fMRI-guided pharmacological blockade, microstimulation, tract-tracing, and computational modeling in non-human primates (NHPs). Work in the lab is organized around Four broad approaches:

First, the use of MRI in humans and NHPs to investigate homologies of brain anatomy and function between these species, to support the applicability of neurophysiology from NHPs to the human case, and to test hypotheses about the fundamental organizational plan of the cerebral cortex in the primate.

Second, the use of well-controlled psychophysics (including longitudinal experiments) combined with microelectrode recording in NHPs to show on a mechanistic level how populations of neurons drive behaviors such as perceptual decisions, categorization, and concept formation and memory.

Third, comparative psychophysical studies in humans and NHPs, as part of a program of neuroethology to understand the relative computational goals of perception/cognition in different primate species. In addition to studies of vision, the lab conducts experiments using auditory and combined audio-visual stimuli, to understand common principles of sensory-cognitive information processing, and to determine how signals across the senses are integrated into a coherent experience.

Fourth, large-scale neurophysiological experiments combined with cutting-edge analysis methods including machine learning, to determine the mechanisms of high-acuity visual perception at the center of gaze. We have developed several eye-trackers that afford photo-receptor resolution, providing an unprecedented look at fine-scale spatial and chromatic processing of the foveal representation in primary visual cortex. 
 

This collaboration will provide the opportunity to work on malignant brain tumors in the laboratories of these three investigators with complementary expertise. The Zhuang laboratory (NIH) has an interest in the investigation of hypoxia (HIF-2a) signaling and tumor development as well as the pathogenesis of brain tumors and development of therapeutics. The Bulstrode laboratory (Cambridge) has an interest in neural stem cell identity and microenvironment interactions in brain development and in brain tumors. The Lathia laboratory (Cleveland Clinic) has an interest in cancer stem cells, tumor microenvironment interactions, immune suppression, and sex differences in brain tumors. The prospective student will have access to mentorship, infrastructure, and resources across all three laboratories. 

Antibiotics are crucial for the treatment of bacterial infections, but they can also cause significant collateral damage to the microbiota (de Nies et al. Nat Rev Microbiol. 2023). Antibiotics supress the growth of commensal microorganisms and at the same time select for drug-resistance. Individuals are often benignly colonized with resistant potential pathogens persisting at low levels within their microbiota, such as extra-intestinal pathogenic Escherichia coli (ExPEC). Antibiotics can lead to overgrowth of these resistant pathogens, facilitating their spread within and between individuals (Stracy et al. Science. 2022).Antibiotics can also lead to resistance genes spreading between bacteria within the microbiota through various horizontal gene transfer (HGT) mechanisms.

This overall aim of this project is to answer the important questions:

1. What are the key factors that determine the level of antibiotic-induced resistance overgrowth/spread within the microbiota?
2. How does antibiotic-induced pathogen overgrowth and level of HGT differ between individuals?
3. Can we engineer the microbiota to prevent the spread of resistance?
    

To achieve this, we will use experimental approaches with synthetic and human-derived microbial communities as well as developing microscopy methods to understand the effect of antibiotics on the micro-scale biogeography of the microbiota. We will test approaches to modify the members of the microbiota to minimize antibiotic-induced pathogen overgrowth or HGT.

Most eukaryotic cells are born with a single centrosome that plays an important part in many aspects of cellular organization. Centrosomes are an excellent model for studying organelle biogenesis because, just like the DNA, they duplicate precisely once during each cell cycle. In the early Drosophila embryo, hundreds of centrosomes synchronously assemble every few minutes as the embryos rapidly progress through repeated cycles of division. 

Although centrosomes are complex nanomachines comprising >400 proteins, only ~10 proteins are absolutely essential for centrosome biogenesis. Thus, to understand the principles that allow these embryos to coordinately assemble so many centrosomes at the right time, in the right place, and then grow them to the right size, we need to understand how these proteins interact with each other, and how these interactions are regulated. This project involves using protein prediction software (e.g. AlphaFold2, Rosetta) to identify putative interactions and then using various approaches (biochemistry, CryoEM, in vitro reconstitution) to validate them (NIH). Once validated, the functional significance of these interactions will be tested using live-cell imaging in the early Drosophila embryo (Oxford). See publications below for two examples of previous collaborations between the NIH and Oxford groups.

Feng et al., Structural basis for mitotic centrosome assembly in flies. Cell, 2017.

Conduit et al., The centrosome-specific phosphorylation of Cnn by Polo/PLK1 drives Cnn scaffold assembly and centrosome maturation. Dev. Cell., 2014.

Radiation (RT) is effective in treating many types of cancers by inducing oxidative stress through the generation of reactive oxygen species (ROS) that result in DNA damage. However, both tumour intrinsic mechanisms for suppressing ROS as well as the hypoxic microenvironment reduce the efficacy of RT. While significant efforts are being pursued to enhance radiation efficacy, our lab will focus on RT-induced ROS that kill cells by inducing ferroptosis. Ferroptosis has recently been described as a non-apoptotic form of cell death dependent on iron and lipid peroxides that contributes to radiation-induced cell death. Studies suggest that the ferroptosis pathway is independent of DSBs and that enhancing ROS induced lipid peroxidation can promote cell killing as well as overcome the problem of tumour radioresistance mediated by intrinsic radical scavengers. NRF2 is a transcription factor playing a major role in protecting cells from oxidative damage. When bound to small MAF proteins, NRF2 transcriptionally activate its target genes through binding to the antioxidative response element (ARE) on their promoter regions. BACH1 is a transcriptional repressor of these antioxidative genes through the competition with NRF2 for ARE and small MAF bindings. In the recent study suggests that NRF2 promotes BACH1-mediated lung cancer metastasis through BACH1 stabilisation. Therefore, there seems to be a tight regulation of NRF2 and BACH1 in promoting cancer through competition or cooperation. In our proposed study we will determine how NRF2 and BACH1 play together in radiation responses while focusing on redox pathways, ferroptosis, and DNA damage.

Patients with neurological diseases often have difficulty in evaluating, planning and initiating actions. This can lead to disorders of motivation, such as apathy and impulsivity. My group studies the cognitive neuroscience of goal-directed action, in the context of disease. Some patients have difficulty evaluating a particular plan in a given context. For example, they might revert to habitual actions which were previously associated with reward, or may be unable to think beyond their current situation. We take a multimodal approach, with behavioural studies, eye tracking, drug studies in healthy people and patients, EEG and neuroimaging. I have also developed computational models – both in the form of abstract cognitive models, and neural network simulations, to understand what the brain computes when determining actions.

My group has found that dopamine plays an important role in energising goal-appropriate actions. We hypothesise that prefrontal goal representations act through corticostriatal loops, being amplified or attenuated by dopamine.  In this project, the student will study patients with Parkinson’s disease and the effects of dopaminergic drugs on cognition, together with neuroimaging (which can include MEG or fMRI), to understand goal-directed action selection and energisation. The student will design their own behavioural tasks to separate out the component processes involved in maintaining and using goal information to energise actions. The student will test patients on their task, test drug mechanisms using a within-subject neuroimaging design, and  deploy appropriate analysis methods with help from a postdoctoral researcher.   The project will also include the opportunity to learn computational modelling if the student is keen.

The WNT signalling pathway regulates patterning and morphogenesis during embryonic development and promotes the renewal of stem cells to maintain tissue homeostasis in adults. Aberrant WNT signalling also drives many types of cancer. Some WNT responses in vertebrates depend on a second signal provided by the R-spondin family of four secreted proteins, RSPO1-4. RSPOs markedly amplify target cell sensitivity to WNT ligands by neutralizing two transmembrane ubiquitin ligases, ZNRF3 and RNF43, which reduce the cell surface levels of WNT receptors. RSPOs can simultaneously engage ZNRF3/RNF43 and the 7-pass transmembrane receptors LGR4, 5 or 6 to trigger the clearance of ZNRF3/RNF43 from the cell surface, followed by lysosomal degradation. RSPO2 and RSPO3 can also engage heparan sulfate proteoglycans (HSPGs) such as glypicans or syndecans to promote ZNRF3/RNF43 clearance in the presence or absence of LGRs. In both cases, ZNRF3/RNF43 clearance results in increased WNT receptor levels at the cell surface and higher sensitivity to WNT ligands.

The molecular mechanism whereby binding of RSPOs to their LGR and/or HSPG receptors and to their ZNRF3/RNF43 effectors promotes the clearance of the trimeric or tetrameric complexes from the cell surface has remained elusive. In this project, students will combine genetic, cell biological, biochemical, biophysical and structural approaches in the laboratories of Dr. Jones at Oxford University and Dr. Lebensohn at the National Cancer Institute to elucidate the underlying mechanisms.

Monoclonal antibodies have emerged in recent years as powerful tools to guide vaccine design and potentially to directly prevent infectious disease. Plasmodium falciparum, which causes malaria, is a relatively unexplored pathogen in this area, with only a few major vaccine candidates dominating the field despite the thousands of proteins expressed by the parasite. This project aims to isolate, characterize and determine the structures of human monoclonal antibodies against known and novel P. falciparum blood-stage antigens using cutting-edge technology. This collaborative project combines the expertise of the Tan lab in isolating human monoclonal antibodies against infectious pathogens and the expertise of the Higgins lab in solving crystal structures of antibody-antigen complexes to identify new sites of vulnerability on parasites at high resolution.