Research Opportunities

Adult neurogenesis, the process of generating new neurons in the adult brain, is a rare occurrence in mammals, confined mainly to the olfactory bulb and the hippocampus. Unlike the body's ability to repair most tissues, the limited scope of neurogenesis in the brain restricts our capacity to recover from neurological damage, a limitation that profoundly impacts the treatment of brain disorders.  In the olfactory system, ongoing neurogenesis supports the regeneration of key neuron types, including dopaminergic cells, granule cells, and olfactory sensory neurons, all of which are essential for sensory processing and adaptability. Recent studies have revealed that dopaminergic neurons born during embryonic development differ significantly from those generated postnatally, suggesting that they perform distinct functions rather than acting as simple replacements.  Our project aims to expand on these findings by exploring whether these differences extend to other regenerating neuron populations in the olfactory system. Using a combination of transgenic mouse models, in vivo calcium imaging, immunohistochemistry, electrophysiology, and behavioral testing, we will investigate the specific roles of embryonic versus adult-born dopaminergic neurons in olfactory processing.  By addressing these questions, our research will contribute to a deeper understanding of the unique contributions of adult neurogenesis to brain function, with the potential to inform new approaches for treating neurological disorders.

The ability to sense and respond to environmental cues is vital for the survival of all organisms. This process hinges on the integration of sensory information to generate appropriate behaviors, a capability rooted in neuronal plasticity. Neuronal plasticity encompasses structural, synaptic, and intrinsic modifications within neurons. However, these mechanisms are often studied in isolation, leaving their collective impact on behavior poorly understood.  Our lab aims to bridge this gap by exploring how mice adapt to olfactory stimuli. In this project, we will manipulate the olfactory environment of mice through sensory deprivation (akin to experiencing a mild cold) or olfactory enrichment (comparable to exposure to a perfume shop). Using advanced genetic tools, we will label neurons responsive to specific odors. Our approach integrates immunohistochemistry, in vivo calcium imaging, and patch-clamp electrophysiology to examine how olfactory bulb neurons adjust their synaptic connections, morphology, and intrinsic properties in response to varying durations of sensory alteration.  To link these cellular changes to behavior, we will employ automated behavioral testing to evaluate the mice's ability to detect and differentiate odors. This will allow us to assess how adaptive plasticity influences learning and behavioral flexibility. By combining cellular and behavioral analyses, this interdisciplinary project aims to uncover the complex neural mechanisms underlying behavioral adaptability in response to changing olfactory environments.

La Crosse virus (LACV), a member of the Bunyaviridae family, is a mosquito-borne pathogen that is leading cause of paediatric encephalitis in North America. Most cases of LACV infection are asymptomatic. However, the virus can cause severe disease when it infects the central nervous system: approximately 1% of LACV neuroinvasive disease cases are fatal, and it causes neurological sequelae including epilepsy and cognitive abnormalities in a further 6-15% of cases. LACV neuroinvasive disease is most common in children under the age of 16.

LACV is an enveloped virus with a tripartite, negative-sense RNA genome consisting of small (S), medium (M), and large (L) segments. The M segment encodes the glycoproteins Gn and Gc that are present on the surface of virus particles. These glycoproteins recognise receptors on the surface of suitable host cells and catalyse the membrane fusion that allows entry of the viral genome into the host cell, the essential first step of the infection cycle. The cell surface receptor(s) of LACV have not been characterised, limiting our understanding of which cells can be infected and of why children but not adults suffer severe LACV neuroinvasive disease.

We have performed an unbiased protein-based screen to identify human cell surface proteins that bind the surface glycoproteins of LACV. We identified that LACV binds to receptors of the Notch signalling pathway. This result is highly significant because Notch receptors are regulators of neuronal development, helping determine whether neural progenitor cells differentiate into neurons or glial cells. Biochemical and biophysical experiments using purified components have confirmed that LACV binds these receptors with high affinity, and we have a high-confidence structural prediction of the interaction between LACV and these cell surface receptors.

This project will define the molecular interactions between LACV and its cell surface receptors at atomic resolution, using a combination of structural biology, biochemical and biophysical techniques. We will exploit this information to generate structurally informed mutations that disrupt these interactions, allowing us to test the functional consequences of Notch receptor binding for LACV infection and neuropathogenesis. Students undertaking this project will gain experience of biochemistry, biophysics, structural biology and molecular virology, with the opportunity to perform virus infection experiments in a high-containment environment. They will also advance our knowledge of an important paediatric disease, potentially identifying new strategies to prevent LACV infection or minimise the subsequent neurological sequelae.

Generation of reactive oxygen species (ROS) by the phagocyte NADPH oxidase is a critical and highly conserved antimicrobial function of myeloid immune cells such as neutrophils and monocytes. ROS production must be tightly regulated to ensure constant readiness for immune defence, while restraining inappropriate activation. A lack of ROS from this complex results in the devastating inborn error of immunity chronic granulomatous disease (CGD), characterised by recurrent infection but also autoinflammation and autoimmunity. Common hypomorphic variation in the genes encoding components of the phagocyte NADPH oxidase also drives pre-disposition to common autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Excess ROS production can, however, result in oxidative stress. Understanding how ROS is tightly regulated is thus important for the development of rational therapeutics immune-mediated diseases. 

Despite the elucidation of the NADPH oxidase complex structure and function, upstream regulators of ROS production remain largely undiscovered due to a lack of robust biological model systems. The Thomas Lab characterised EROS (Essential for Reactive Oxygen Species) as an indispensable regulator of ROS generation but we believe that there are many more. Recent developments in CRISPR-Cas9 technology now allows both the introduction of precise edits (homology-directed repair, HDR) and genome-wide forward genetic screening by introducing knockout (CRISPRko) libraries. This may identify therapeutic targets in inflammatory disease.

We will use CRISPR-HDR methods to endogenously tag key components of the NADPH oxidase complex with fluorescent proteins to generate reporter lines for iterative selection by flow cytometry. By screening these at genome-wide scale with CRISPRko libraries and sorting cells based on component expression, followed by functional screens using fluorescent ROS probes, we will elucidate upstream regulators of the complex expression and function. The function of these novel regulators can then be investigated and validated using primary and immortalised cells, structural biology, and selective mutagenesis. Interrogation of publicly available genomic datasets will guide ‘hit’ selection and possible therapeutic relevance.

The placenta is the interface between mother and fetus, integrating signals between the two. The placenta releases factors such as proteins and miRNAs that can impact on maternal and fetal physiology. Some of these will be released from the placenta within extra-cellular vesicles (EVs), but how their content is modulated by an obese diabetic environment and how it impacts on maternal and offspring health is unknown. 

This project will explore how the placental secretome, including EV content, is modulated by obesity and diabetes during pregnancy and define how these changes have short and long-term consequences on maternal and offspring metabolism. The project will involve:
(1) profiling the placental secretome in healthy and obese diabetic pregnancies
(2) determining the protein and miRNA content of placental EVs isolated from lean and obese diabetic murine pregnancies
(3) establishing the effects of secreted placental proteins on maternal and offspring metabolism
(4) a combination of in vitro and in vivo experiments to establish the functional consequences of the changes in placental EV protein and miRNA content 

Lymphocytes respond to infection by rapidly increasing and decreasing the expression of many genes in a highly regulated manner. This regulation requires the integration of transcription, mRNA decay and translation. We are only just beginning to understand how these processes are integrated with each other. The host labs are studying how the multiprotein CCR4-NOT complex and its associated RNA binding proteins control gene expression. By combining structural and molecular biology approaches with cellular immunology and mouse models of immune responses we offer a broad training experience and the opportunity to discover fundamental mechanisms of gene regulation in the immune system. The discoveries have application in modifying the potency and durability of cellular immune therapies such as anti-tumour CART cell responses and the student will have an opportunity to apply fundamental knowledge to these applications.

References
The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins. Nat Commun. 2022 doi: 10.1038/s41467-022-29979-x. PMID: 35477960; PMCID: PMC9046422.

Regulation of the multisubunit CCR4-NOT deadenylase in the initiation of mRNA degradation. Curr Opin Struct Biol. 2022 doi: 10.1016/j.sbi.2022.102460. Epub 2022 Sep 16. PMID: 36116370; PMCID: PMC9771892.

The nexus between RNA-binding proteins and their effectors. Nat Rev Genet. 2023. doi: 10.1038/s41576-022-00550-0. Epub 2022 Nov 23. PMID: 36418462; PMCID: PMC10714665.

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.