Research Opportunities

The human placenta is the first organ of the embryo and it is functional immediately after implantation. Before fetal organogenesis, the placenta holds a multi-functional and unique role as a physical, chemical, and cellular barrier. It alone orchestrates the chemical communication between mother and fetus. Most of this communication is mediated by the secretion of specific placental peptide hormones (hCG, hPL, etc.) into the maternal bloodstream. Despite these hormones having developmental and irreplaceable functions, little is known about their intracellular life: synthesis, intracellular traffic, secretion, and degradation. More importantly, many pregnancy disorders are associated with lower expression and levels of these hormones in circulation, such as fetal growth restriction and preterm birth. The cell biology of these diseases is not well understood.

With the recent advance in human placenta organoid development and novel culture techniques of trophoblasts derived from stem cells, we can now finally interrogate the fundamental questions about placental hormones' intra and extra-cellular fate. In combination with the diverse set of advanced imagining methods available between the co-mentors of this project (advanced fluorescence and cryo-electron microscopy), innovative multi-omics techniques, and advanced biochemistry and cell biology approaches, we propose to 1) discover the diversity of secretory granules (SGs) expressed in and secreted from the human placenta; 2) implement a novel in vitro secretomics approach to determine the molecular machinery that regulates the secretion of the SGs and their content; 3) validate the mechanisms using human placenta and isolated trophoblasts from donated tissue and new placenta organoids culture.

The successful candidate will have the opportunity to train on several modern imaging techniques and learn about the fundamentals of placenta development and physiology while using a multi-disciplinary approach in a team of expert cell biologists from both institutions and generating impactful basic and applied research.

Maternal over-nutrition and obesity during pregnancy is known to have long-term effects on the health of the offspring, including increased risk of obesity. Weight gain in offspring exposed to maternal over-nutrition is at least in part caused by hyperphagia- implicating altered function of hypothalamic energy homeostatic pathways as an underlying cause- but the precise mechanisms by which the in utero environment impacts on hypothalamic development is unclear. Key metabolic hormones such as insulin, leptin and ghrelin have a dual role during brain development as growth factors. These metabolic hormones are altered in an obese pregnancy, providing a direct route by which the maternal nutritional state can impact on offspring hypothalamic development. We will use a combination of in vivo manipulation of hormone levels (e.g. fetal brain injection) and ex vivo neuro-developmental techniques (e.g. neurospheres) to examine the consequences of altered metabolic hormone levels for early hypothalamic development. We will also use immunofluorescence and viral tracing to study hypothalamic architecture in the offspring of obese mothers once they reach adulthood, and correlate the anatomy with functional readouts of complex feeding behaviours using operant and metabolic chambers.

Mitochondrial diseases are caused by defects in genes required for energy production and oxidative phosphorylation (OxPhos). We find it intriguing that some patients with mitochondrial disease present late in life, with very tissue-specific phenotypes. It seems that not all cells and tissues are equally susceptible to mitochondrial disease.

We mainly study how mitochondrial dysfunction and mutations in the mitochondrial genome affect neural stem cell behaviour in Drosophila and mouse. The questions we address are:
(1) how mitochondrial dysfunction affects normal and pathological cell fate decisions in the developing brain. We previously showed that neural stem cells in the brain rely heavily on mitochondrial energy production and now study how they interact with the glial cells that make up their stem cell niche.
(2) how transcription of the nuclear genome is regulated when a cell is confronted with mitochondrial dysfunction. We employ and develop innovative DamID-based in vivo chromatin profiling technology to study metabolism of chromatin modification.
(3) how mutations in the mitochondrial genome evolve over time, during brain development and aging. We use in situ hybridisation-based methods and single-cell CRISPR screening to identify novel regulators of mitochondrial genome maintenance.

In order to study these questions in an in vivo context, in (stem) cells surrounded by their appropriate tissue environment, our primary model system is the fruit fly, Drosophila melanogaster. In addition, we actively translate our findings and the technology we develop into mammalian model systems, in particular the mouse embryonic cortex.

Relevant references
- van den Ameele J, Krautz R, Cheetham SW, et al., Reduced chromatin accessibility correlates with resistance to Notch activation. Nat Commun. 2022;13(1):2210.
- van den Ameele J, Li AYZ, Ma H, Chinnery PF. Mitochondrial heteroplasmy beyond the oocyte bottleneck. Semin Cell Dev Biol. 2020 Jan. 97:156-66.
- van den Ameele J, Brand AH. Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation. eLife. 2019;8:e47887.
- Tiberi L*, van den Ameele J*, Dimidschstein J, Piccirilli J, Gall D, Herpoel A, Bilheu A, Bonnefont J, Iacovino M, Kyba M, Bouschet T, Vanderhaeghen P. Bcl6 induces neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets. Nat Neurosci. 2012 Dec;15(12):1627-35.
- Gaspard N, Bouschet T, Hourez R, Dimidschstein J, Naeije G, van den Ameele J, Espuny-Camacho I, Herpoel A, Passante L, Schiffmann SN, Gaillard A, Vanderhaeghen P. An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature. 2008 Sep 18;455(7211):351-7.

The presence and role of neural stem cells (NSCs) in the adult human brain is a long-debated issue in neuroscience. Recent work has demonstrated that stem-like cells exist in the embryonic, foetal, and human adult brain where they persist well into adulthood and can even contribute to neurogenesis. However, their role in neurodegenerative disease is unknown. Ongoing work in the lab has led to the hypothesis that NSCs may become dysfunctional in neurodegenerative disease resulting in senescence chronic inflammation, and thereby acting as pacemaker cells driving neuronal demise. This ambitious project aims to identify disease-associated NSCs and their phenotype in the context of human neurodegeneration using spatial biology approaches, including imaging mass cytometry, RNA scope and single nuclear RNA sequencing. Relying on post-mortem brain tissue of different stages of Alzheimer’s disease, traumatic brain injury, vascular dementia and chronic stroke, this project will study NSCs in a range of human diseases characterised by neurodegeneration and neuronal injury. Ongoing work in the lab identifies NSC-specific markers based on transcriptomics and protein profiling experiments in brains with progressive multiple sclerosis, enabling to investigate the distribution of NSCs in a wide range of diseases. Spatial transcriptomics and proteomic approaches will allow to study their phenotype and dysfunction in relation to other cell types and local pathology. This project will shed light on the role of NSCs in neurodegeneration and has the potential to identify an entirely novel mechanism of neurodegeneration in human disease.

This project will be co-supervised by Prof. A. Quaegebeur.

Multipotent self-renewing hematopoietic stem cells (HSCs) support life-long blood system homeostasis and play essential roles in human disease and its therapy. HSC transplantation is an important cell therapy for a range of hematological diseases including immunodeficiencies, beta-globinopathies, and blood cancers. Through their ability for self-renewal and multipotency, HSCs can reconstitute the hematopoietic system following transplantation. Most HSC transplants are performed using allogeneic HSCs but there is also a growing interest in the development and use of autologous HSC transplantation gene therapies for a range of non-malignant blood diseases. A major unresolved question in the field is what regulates the fitness of an HSC. High fitness HSCs display durable and balanced blood system reconstitution activities. By contrast, low fitness HSCs have weak or biased activities. The accumulation of low fitness HSCs is thought to contribute to various disease pathologies and their use in HSC transplantation can result in engraftment failure. Building on research interests in the Muljo lab at the NIH and the Wilkinson lab at the University of Oxford, this project will focus on characterizing transcriptional and post-transcriptional mechanisms regulating HSC fitness. Biological mechanisms identified here will be used to devise new strategies to enhance life-long hematopoietic system health and to improve the safety and efficacy of HSC transplantation therapies.
 

Recent publications:

Wang, S., Chim, B., Su, Y., Khil, P., Wong, M., Wang, X., Foroushani, A., Smith, P. T., Liu, X., Li, R., Ganesan, S., Kanellopoulou, C., Hafner, M. and S. A. Muljo. Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3. Genes & Development, 33: 1048–1068. DOI: 10.1101/gad.325100.119.

Wilkinson, A.C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R.V., Yamamoto, R., Loh, K.M., Nakamura, Y., Watanabe, M., Nakauchi, H. and S. Yamazaki. (2019). Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature, 571: 117–121. DOI: 10.1038/s41586-019-1244-x.

Haney, M.S., Shankar, A., Hsu, I., Miyauchi, M., Palovics, R., Khoo, H.M., Igarashi, K.J., Bhadury, J., Munson, C., Mack, P.K., Tan, T., Wyss-Coray, T., Nakauchi, H., Wilkinson, A.C. Large-scale in vivo CRISPR screens identify SAGA complex members as a key regulators of HSC lineage commitment and aging. bioRxiv 2022. DOI: 10.1101/2022.07.22.501030

Immunity and immune-tolerance at mucosal and other barrier surfaces is vital for host survival and homeostasis with antibodies or immunoglobulins (Ig) playing a key role. However, the specific roles of B cell sub-types, particularly, the innate-like B-1 cell subset is poorly understood. The surgeon James Rutherford Morrison (1906) called the omentum in the peritoneal cavity the "abdominal policeman" and it promotes gut IgA production by peritoneal B-1 cells. IgA is the most abundantly produced antibody isotype and is known to be important for mucosal immunity. It is estimated that ~50% of IgA is derived from B-1 cells. In addition, B-1 cells are thought to be important because they make T cell-independent “natural” IgM circulating in our blood, and they can rapidly respond to mucosal perturbations such as an infection. By contrast, it will take conventional B-2 cells weeks to mount a germinal center (GC) reaction and generate antibodies that have undergone T cell-dependent affinity maturation and isotype switching. Textbooks currently do not entertain the possibility that B-1 cells can also participate in GC reactions. This project aims to challenge such an assumption. After all, B-1 cells in the gut mucosa and probably other mucosal tissues can undergo class switch recombination to IgA. However, the differentiation program that leads to this distinct pathway of IgA production is not well understood: for example, it is unknown if this process occurs outside or within GCs in mucosa-associated lymphoid tissues (MALT). Expertise on B-1 cells in the Muljo lab and the GC reaction in the Turner lab will be combined to explore this potentially paradigm-shifting research. Both wet-bench and bioinformatic research opportunities are available.

Students will learn about the fundamentals of transcriptional; epigenetic and post-transcriptional regulation; immunometabolism; in vivo CRISPR screening; CRISPR editing in primary B cells; and systems immunology. The combination of classical immunological techniques and cutting-edge, multi-disciplinary approaches will enable important discoveries to define the in vivo biology of B-1 cells. Ultimately, we seek novel insights that can be translated to inform vaccine design targeted to activate B-1 cells and/or therapeutics to inhibit their activity when necessary.
 

Recent publications:

Turner, D. J., Saveliev, A., Salerno, F., Matheson, L. S., Screen, M., Lawson, H., Wotherspoon, D., Kranc, K. R., and Turner, M. (2022). A functional screen of RNA binding proteins identifies genes that promote or limit the accumulation of CD138+ plasma cells. eLife, 11, e72313. PMID: 35451955; DOI: 10.7554/eLife.72313.

Osma-Garcia, I.C., Capitan-Sobrino, D., Mouysset, M., Bell, S.E., Lebeurrier, M., Turner, M. and Diaz-Muñoz, M.D. (2021). The RNA-binding protein HuR is required for maintenance of the germinal centre response. Nature Communications, 12(1):6556. PMID: 34772950; DOI: 10.1038/s41467-021-26908-2.

Wang, S., Chim, B., Su, Y., Khil, P., Wong, M., Wang, X., Foroushani, A., Smith, P. T., Liu, X., Li, R., Ganesan, S., Kanellopoulou, C., Hafner, M. and S. A. Muljo. (2109). Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3. Genes & Development, 33: 1048-1068. PMID: 31221665; DOI: 10.1101/gad.325100.119.

The Rayon’s lab (www.rayonlab.org) overall aim at the Babraham Institute in Cambridge is to investigate the molecular and metabolic pathways that control biological timing and lifespan. To answer these questions, we work with mouse and human stem cells and embryos and employ a variety of quantitative and genomic techniques. We are looking for applicants that are curious about evolution, developmental biology and embryonic stem cells.

We are interested in the following topics:
1.    Understanding the regulation of enhancer timing. We want to test the existence of species-specific enhancers and their dynamics.
2.    Exploiting genetic variation to investigate dynamics of regulatory networks in stem cell models.  
3.    To develop a high-content imaging assay to screen for modulators of timing. Develop a screen to explore if epigenetic, metabolic, and turnover factors impact the pace of differentiation.

A background on cell culture or molecular biology, as well as skills in bioinformatics or computational approaches would be useful, but ample opportunities for training will be provided.

Virus infection of brain stem cells represents a major global health concern, but also offers treatment possibilities in neurodegenerative diseases and in malignant brain tumours. For example, Zika Virus targets SOX2+ neural progenitors in the developing brain to cause microcephaly in babies born to mothers infected during pregnancy; likewise, it targets SOX2+ glioma stem cells in the most common and lethal malignant brain tumour, glioblastoma (GBM) (https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4135719). Stem cells have been shown to exploit a distinct set of antiviral mechanisms compared to somatic cells (https://doi.org/10.1016/j.cell.2017.11.018), and viral permissivity varies widely between example developing brain and glioblastoma stem cell populations. Understanding and manipulating the cell-intrinsic mechanisms underpinning antiviral resistance in brain stem cells will inform approaches to protect and exploit neural stem cell function and to ablate cancer stem cells in GBM.

This project will seek to understand and modify viral permissivity and antiviral defence mechanisms in developing brain and brain tumours, focusing on stem cell intrinsic pathways. In the first place we will  address the hypothesis that immune selection pressure on glioma stem cells during tumour development results in expansion of a mesenchymal/injury-response cell population (https://www.cell.com/cell/pdf/S0092-8674(21)00351-2.pdf) refractory to virus infection, then proceed to examine underlying mechanisms.

You will be working with human patient-derived and mouse defined mutation glioma cell models, treated with viruses and viral mimetic compounds. You will be assaying transcriptional identity and responses using qPCR and RNA sequencing, immunofluorescence and RNA smFISH, and cytokine secretion using immunoassays. The lab has access to the state-of-the-art Cambridge Stem Cell Institute imaging, FACS, sequencing and bioinformatic facilities, and to additional specialist facilities (e.g. imaging mass cytometry) in the CRUK Cancer Institute next door. We will validate results in mice in vivo and/or slice cultures prepared from  human developing brain and patient brain tumour tissue, models we have established and use routinely in the lab.

The bacterial cell envelope comprises the cell wall and either one or two membranes. The cell envelope is of major interest in infection biology because it is the site at which pathogenic bacteria interact with their host organism. In particular, bacterial virulence proteins must be transported across the cell envelope to affect the host.

We aim to understand the molecular mechanisms by which proteins, nucleic acids, and mechanical force are transferred across and along the cell envelope in processes of biomedical importance. The project is to undertake structure-led studies of  the dedicated nanomachines that carry out these processes. The NCI part of the collaboration will involve structural analysis, primarily by cryoEM. The Oxford part of the collaboration will concentrate on complementary mechanistic work using biochemical, genetic, and live cell single molecule imaging methods. Our groups  have collaborated for more than 15 years on projects including the transport of folded proteins across the bacterial inner and outer membranes (Tat protein transport system and Type 9 Secretion System), lipoprotein export, DNA transport during horizontal gene transfer (conjugation and natural competence), and gliding motility.

Our group is interested in uncovering and understanding key mechanisms of disease that affect cardiac muscle function. We have a particular interest in understanding how regulation of cardiac muscle contraction is altered in common acquired and inherited cardiovascular diseases. We do this by using cutting edge techniques in cellular imaging, employing human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), and CRISPR/Cas-9 to understand human cardiovascular disease in the dish.
 

We have two key focuses in the lab:

1. Understanding how inherited heart conditions including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) alter cardiomyocyte function. We do this by using CRISPR Cas-9 genome engineering in combination with iPSC-CMs to screen how HCM and DCM causing variants cause disease. We can then use these systems to screen novel therapeutics in the dish. We have designed multiple techniques to make these analyses feasible and rapidly deployable: SarcTrack and CalTrack.
    
2.  Investigate the processes that alter cardiac muscle function in acquired cardiac diseases including myocardial infarction, atrial fibrillation, and heart failure. We are able to use biochemical techniques twinned with fluorescent imaging to assess how cardiac myosin states are altered in disease tissues. This technology allows us to uncover key disease mechanisms that alter heart muscle function, allowing insight into these common heart muscle diseases. 
    

We have multiple key collaborations within the University of Oxford and internationally. Together we focus on pushing the boundaries of understanding in acquired and inherited cardiovascular disorders of the heart muscle. Within the group we have an inclusive and diverse set of researchers who have a wide range of cutting-edge expertise. Within the wider lab environment and our collaboration network we have over 20 researchers in this area.
 

Scientific training opportunities in the lab include but are not limited to:
 

1. Induced pluripotent stem cell culture
2. Techniques for iPSC to cardiomyocyte differentiation
3. CRISPR/Cas-9 genome engineering
4. PCR and genetic sequencing
5. A wide range of standard fluorescent microscopy and confocal microscopy
6. Phenotyping of cardiomyocyte function with fluorescent probes and genetically encoded protein tags
7. RNA sequencing and qPCR
8. Western blotting and phosphoprotein blotting techniques
9. Drug screening using live cell microscopy

Transferrable skills include but are not limited to:
 

1. Learning to interact with MatLab and other computing packages for automating and simplifying data analysis.
2. Using genome viewers and associated software for designing and executing CRISPR/Cas-9 engineering.

References:

1.  Hypertrophic cardiomyopathy mutations in MYBPC3 dysregulate myosin Science Translational Medicine 2019
2. Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy Circulation 2020
3. SarcTrack An Adaptable Software Tool for Efficient Large-Scale Analysis of Sarcomere Function in hiPSC-Cardiomyocytes Circulation Research 2019
4. CalTrack: High-Throughput Automated Calcium Transient Analysis in Cardiomyocytes Circulation Research 2021
5. Common genetic variants and modifiable risk factors underpin hypertrophic cardiomyopathy susceptibility and expressivity Nature Genetics 2021

Retroviruses, flaviviruses and coronaviruses are important pathogens capable of causing serious human disease. To replicate and disseminate infection, these viruses hijack host cell machinery, including organelles such as the endoplasmic reticulum, the Golgi apparatus, lipid droplets and the nucleus to generate progeny virions. Sensors within the innate immune system detect virus-induced membrane reorganization, identify virus weak spots, and influence disease progression in this complex virus-host interplay. This project focuses on the innate immune response that specifically targets virus replication and translation complexes (RTCs) anchored at the membrane of cell host organelles. RTCs isolated from these sites will be studied using molecular and cell biology, in combination with bioinformatics, structural biology and high resolution imaging to design novel antiviral interventions.

The laboratory investigates the interactions between episodic memory formation and brain dynamics during sleep. How are memories strengthened (‘consolidated’) overnight? What is the role of specific brain rhythms during sleep? Can we use sleep to experimentally control the extent of forgetting? To tackle these questions, a range of techniques including intracranial EEG, scalp EEG, MEG, fMRI, non-invasive brain stimulation (NIBS), and behavioural testing are used.

Multisensory learning helps an individual learn through more than one sense. However, the underlying neural mechanism is unclear. In this study we aim to pursue this question in a spatial learning regime. We will focus on the medial entorhinal cortex (MEC), which plays a critical role in spatial learning and the dysfunction of which is closely related to Alzheimer’s disease. We will record neural dynamics of the MEC using two-photon imaging approach when mice navigate in virtual environments, in which multisensory spatial information will be precisely delivered. The goal of the project is to deeply understand how the neural response of the MEC contributes to multisensory learning.

The formation of infectious HIV-1 particles requires the incorporation of Env glycoproteins during the assembly process, with Env mediating binding of newly released virions to target cells and subsequent entry via fusion between viral and target-cell membranes. The interaction between the MA domain of Gag and gp41 of Env plays a central role in Env incorporation into virions and in the activation of Env fusion activity, yet this interaction remains poorly understood at the biochemical and structural levels. In addition, recent structures of the MA lattice in both immature and mature HIV-1 particles implicate a maturation-induced rearrangement of MA the lattice. This opens the possibility that MA-gp41 interactions are dynamic, and change during the maturation process.

We aim to apply an array of biochemical, structural, super-resolution imaging, and virological approaches that will interrogate the MA/gp41 interaction and will provide novel insights into the mechanism and dynamics of Env incorporation into HIV-1 particles. A set of informative mutants, which display stabilized MA-MA and/or MA-gp41 interactions, will be used for structural studies,. Cryo-ET analysis of intact mature and immature virions will be performed to obtain structural data that will complement the information obtained with the MA/Gag assemblies. Predictions regarding potential sites of MA-MA and MA-gp41 interactions obtained from these structural studies will be tested in Env incorporation and virus replication assays. This combination of structural, biochemical, and virological studies will help to elucidate the mechanism of Env incorporation into HIV-1 particles and the subsequent process of particle maturation.

Human immunodeficiency virus type 1 (HIV-1) is the causative agent behind acquired immunodeficiency syndrome (AIDS) that currently has no cure or vaccine. While antiviral treatments are effective, the rise of drug-resistant strains has become a growing concern. HIV-1 primarily infects the immune system, targeting CD4+ T cells and macrophages and is a lentivirus known to be able to infect non-dividing cells, requiring it to exploit nuclear import mechanisms. This process is dependent on the viral capsid. The HIV capsid is a conical structure that houses the genomic material of the virus. It needs to be metastable in order to be protective while allowing timely disassembly (termed uncoating) to release its genome. The dynamics of the capsid nuclear import and uncoating are still unknown and are modulated by host dependency and restriction factors.

We aim to apply multi-imaging modalities to investigate uncoating and nuclear import of HIV. These will include super-resolution fluorescence microscopy (including the newest MINFLUX technology), Focused Ion Beam and Scanning electron microscopy (cryoFIB/SEM), cryo-electron microscopy and cryo-electron tomography (cryoEM/ET). The viral core and host factors will be fluorescently tagged, and infection will be monitored from viral attachment to nuclear import. The sample will be cryo-preserved and imaged by cryoEM/ET and cryoFIB/SEM. The combination of these imaging techniques, paired with molecular biology and virology tools, will yield unparalleled knowledge of the HIV infection process within the native cells, providing the framework for development of novel therapeutics targeting HIV infection in the future.