Cells use lipids as blocks to build their membranes. Therefore, regulation of lipid synthesis is essential for proper cell growth and proliferation. In addition, the carefully orchestrated production of lipids during development underlies striking morphological changes and functional specialization in a variety of cell types such as neurons or antibody secreting cells. In this project we want to investigate how cells control the levels of phosphatidylcholine (PC), the most abundant lipid component of eukaryotic cell membranes. Although the pathways that generate PC have been well described, how cells sense and adjust the levels of PC within their membranes remain still poorly defined. Understanding these mechanisms is clinically important because PC deficiency is linked to several human diseases like lipodystrophy, fatty liver, retinal dystrophy and short stature.

The enzyme that controls the rate of PC production is called Pcyt1a. Because PC is a cylindrical lipid, its depletion disrupts proper lipid packing within the membrane; this is sensed by Pcyt1a, which is then activated to correct the defect. Our recent work has shown that this process takes place inside the nucleus of many different eukaryotic cells. This finding is surprising given that lipid synthesis is mostly a cytoplasmic process. By using a combination of state-of-the-art live cell imaging, genetic and biochemical approaches both in eukaryotic model organisms and mammalian cells, the project aims to:

1. Investigate the molecular mechanisms by which Pcyt1a activity inside the nucleus coordinates PC sensing with the production of membranes that are used for organelle and cell growth.
2. Elucidate the cellular and physiological consequences of human disease-linked Pcyt1a mutations which are known to cause distinct biological phenotypes.

Intracellular transport is driven by nanoscale motor proteins that use energy derived from ATP hydrolysis to move cargo between different cellular compartments along cytoskeletal tracks. Myosin motor proteins perform a wide range of fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Myosin motor activity control within the cellular environment involves on/off switches, however, it remains unknown whether myosin motors can regulate their speed or processivity and fine tune their activity to a specific cellular task. Several classes of myosins are expressed simultaneously in eukaryotic cells and thus complex regulatory mechanisms are in place to control the precise spatial and temporal activation or inhibition of myosin motors at the cellular level.

The focus of this PhD project is to determine the mechanisms that regulate myosin motor activity and cargo attachment. The key questions are to:
• Analyse whether myosin properties are regulated by phosphorylation in the motor domain
• Investigate the impact of actin filament track composition on myosin activity
• Determine the molecular signals that trigger cargo attachment and release

In this project the student will use a wide variety of cell biological, biochemical and biophysical techniques including phospho-proteomics, in situ proximity labelling, super resolution live cell imaging, expression and purification of proteins, in vitro motility assays, stopped-flow experiments, kinase assays and a wide variety of imaging techniques.

This fundamental research into the mechanisms of myosin regulation and their cellular functions in different cell types and tissues is significant and important, because defects in myosin activity are linked to a wide range of diseases. Myosin motors are established drug targets and therefore a detailed understanding of the molecular mechanisms that regulate myosin activity and their cellular activities are essential to design new approaches for successful treatment.

The brain is a key player in the control of appetite, metabolism and endocrine functions.
Our lab studies pathways through which the brain senses and integrates nutritional and metabolic signals in the regulation of energy and glucose homeostasis. We have a specific interest in the brain pathways involved in protein sensing and the regulation of satiety and hunger.

It is well known that protein can potently suppress hunger. Less is known about the effects of protein restriction on appetite, but data suggest that animals fed a low-protein diet overeat. The underpinning mechanisms are unknown. Understanding this in the wider framework of homeostatic regulations might reveal brain circuits responsible for a “proteostat” and significantly increase our understanding of brain pathways regulating appetite.

Using the modern neuroscience toolbox, we will identify and characterise the cellular, molecular and neuroanatomical attributes of brain cells involved in the detection of protein deficiency and mediating the associated hyperphagic response. This project will provide the opportunity to learn a wide variety of in vivo neuroscience techniques, including TRAPPING, chemogenetic, fiber photometry, circuit mapping and various histology techniques including immunofluorescence, electron microscopy and light-sheet imaging of cleared whole brains. The student will work in partnership with a postdoc on this project, with Dr Blouet, as well with key technical staffs for more specialised expertise.

The student will also benefit from our bespoke training programme and will develop skills in critical appraisal, research design, data management, transparency, presentation skills and scientific publishing.

This would be an ideal project for someone interested in understanding how dietary signals are sensed by the brain to regulate appetite and metabolism, with links to the pathophysiology of obesity and metabolic diseases.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative
diseases that are difficult to diagnose and with no effective treatment available. Among various
ALS subtypes, a large proportion is linked to TDP43 with a high degree of pathogenic overlap with
other ALS and FTD associated genes (such as FUS). An RNA/DNA-binding protein, TDP43 has
multi-faceted roles in cellular homeostasis and survival. While located predominantly in the nucleus,
it shuttles between various cellular compartments using its nuclear localization and nuclear export
sequences. Loss of normal TDP43 function leads to nucleo-cytoplasmic translocation and
aggregation, hallmarks of disease initiation. Over the years, several mechanisms have been proposed
for TDP43 pathogenicity including dysregulation of mRNA processing, nucleo-cytoplasmic
mislocalization/aggregation as well as abnormal stress responses due to its association with stress
granules. However, study of these protein- and/or RNA-linked disease mechanisms have failed to
lead to disease modifying therapies.
Recent evidence started to emerge for GIN as an important contribution in ALS and FTD. At
close inspection it has been shown that in neurons TDP43 accumulates at toxic double strand breaks
(DSBs) where it interacts with other DDR genes to promote DNA-repair via a process called non-
homologous end-joining (NHEJ). That being said is not very clear what type of endogenous
stress is responsible for the build-up of DSBs and what is the exact mechanism of action. We have
shown in the past that members of the NHEJ pathway behave differently depending of the replication
status and cell types starting to point more and more to a re-wiring of DDR in neurons as compared
to replicating cell types. This requires for comparative analyses between replicating vs. non-
replicating neurons in TDP43 and FUS deficient backgrounds as paralleled to the normal wild-type
scenario. Further investigation along these lines may add new dimensions to our knowledge of
genome repair and their defects in neurodegenerative diseases and allow us to develop clinically
effective strategies to ameliorate genome instability in ALS-TDP-43.

Microglia, the brain’s resident immune cells, play a key role in brain development, learning and memory, as well as in the behavioural adaptation to various environmental challenges (like stress, inadequate nutrition, and viral infections), which have been linked to the development of depression, schizophrenia, and autism spectrum disorders (ASDs).
‘Dark microglia’ is a newly described microglia phenotype that is rarely present under steady state conditions. Rather, dark microglia become abundant in development, chronic stress, aging, and neurodegeneration, where they play a major role in the remodelling of neuronal circuits, especially at synapses.
This PhD project will investigate the role and function of microglia and dark microglia in normal development and following experimental maternal immune activation (MIA) in wild type and genetically modified laboratory animals with dysfunctional dark microglia.
Combining disease modelling, behavioural and histopathological analyses, with cellular phenotyping and spatial proteomics, this project aims at providing new molecular insights into the function of microglia in development, which will help generating new strategies to target innate immune function in neurodevelopmental disorders.

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. Applicants or rotation students will be involved in addressing one of the following questions:
(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.

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 growing prevalence of obesity worldwide means that in many populations at least 50% of women are overweight or obese at the start of pregnancy. Obesity is a major risk factor for gestational diabetes mellitus (GDM), which affects approximately 1 in 7 pregnancies globally. Metformin, a biguanide drug, effectively controls maternal glycaemia in humans and is a first line drug therapy for GDM in many countries. However, metformin freely crosses the placenta and rapidly reaches similar levels in the fetal circulation as the mother. We have shown that metformin rapidly enters the placenta and fetal tissues including the brain. Metformin therefore has the potential to have direct effects on placental and fetal tissues, independently of changes in maternal metabolism. We have shown that metformin alters cellular metabolism in the placenta, including causing a in a reduction in ATP production which and is associated with increased adiposity postnatally. The trajectory of effects on offspring adiposity display sexual dimorphism. In adult animals, Metformin has been shown to alter signalling in the hypothalamus and reduce body weight. However, how Metformin affects the fetal hypothalamus- and whether Metformin- induced changes in hypothalamic energy balance are an underlying cause of increased adiposity in exposed individuals- remains unknown. This project will investigate the impact of fetal Metformin exposure on cellular metabolism in the hypothalamus, and how this alters hypothalamic development. We will combine these experiments with long-term studies of feeding behaviour in in utero Metformin- exposed animals to investigate whether altered hypothalamic feeding control is an underlying cause of the previously reported increased adiposity.

Gut hormones control post-prandial metabolism and appetite and have been harnessed for some very successful new treatments for type 2 diabetes and obesity, based on the hormone GLP-1 (glucagon-like peptide-1). Our group is interested in how gut hormones like GLP-1 are released from the gut, and how they act on target tissues.
To enable research into the rare enteroendocrine cells that produce gut hormones, we genetically engineer human intestinal organoids to express fluorescent sensors under the control of cell-specific hormonal promoters. These organoid lines allow us to identify and perform dynamic cell imaging and electrophysiology of identified cell types in the human gut, which when combined with results of RNA sequencing are used investigate molecular signalling pathways underlying responses to different nutritional and pharmacological stimuli at the single cell level.
This project will investigate the roles of signalling pathways involving cyclic AMP and ERK in GLP-1 producing L-cells, and their importance for stimulus detection and exocytosis in L-cells. We will perform live cell imaging of cAMP, calcium and pERK in human L-cells in response to different stimuli, after generating new organoid lines by CRISPR-Cas9 expressing genetically encoded fluorescent sensors for these different signalling pathways. The importance of candidate regulatory proteins will be examined by generating gene knockouts by CRISPR-Cas9, and effects on hormone secretion will be measured by immunoassay and peptide mass spectrometry.
Overall, we aim to develop new treatments for metabolic disease based on targeting the gut endocrine system, which requires a deeper understanding of different signalling pathways in enteroendocrine cells.

Maternal obesity during pregnancy is associated with increased risk of poor cardiometabolic health in the offspring. Although the role of miRNAs (a family of small non-coding RNAs) in health and disease and interest in miRNA-based therapeutics are both gaining increasing interest, the potential role of programmed miRNAs as mediators of a suboptimal in utero environment on long-term health has been largely ignored. We recently observed dysregulation of abundant cardiac miRNAs in the hearts of ageing mice that were exposed to maternal obesity whilst in utero. This project aims to establish how these programmed miRNAs contribute mechanistically to programmed cardiac dysfunction. The PhD student will determine if these miRNAs are changed during different stages of development in mice exposed to maternal obesity and establish if their expression is modulated by a post weaning obesogenic diet. The student will use in vitro and in vivo gain/loss-of-function experiments to determine if these miRNAs have a role in programming of the cardiac phenotype. This project will ultimately contribute to our understanding of how in utero exposure to obesity impacts on our long-term cardiovascular health and establish the potential use of miRNAs as biomarkers of programmed disease risk.

The importance of circadian medicine for human health is rapidly becoming apparent. Humans have an intrinsic circadian clock that synchronises physiology with the external environment and controls the expression of up to 40% of protein coding genes. Most clinical advances in circadian medicine have been focussed on chronotherapeutics and drug dosing which have been the traditional research areas for human chronobiologists. However, 52% of genes associated with rare disease are predicted to oscillate across the course of a day, thus there is an unexplored potential for the role of the circadian clock in the study and treatment of rare disease, and the contribution to diseases with diurnally variable pathophysiology. You will contribute to our ongoing project to pilot the idea of Chronomic Medicine – merging chronobiology and genomic medicine, by exploring the mechanistic basis for the role of the circadian clock in the inheritance and phenotypes of Inherited Cardiac Arrhythmia (ICA). We have identified a set of enriched variants in the 100,000 genomes project ICA cohort located in circadian motifs upstream of rhythmic disease associated genes. One such example is a two variant haplotype in circadian E-box motifs upstream of SCN5A that has previously been thought to modulate arrhythmias, but the mechanism remains unknown. We are establishing the contribution of the two E-box motifs that contain the conserved haplotype to the circadian regulation of the promoter in this canonical arrhythmia gene. To perform functional genomics on these variants we are taking a three-fold approach, using gene editing, methylation analysis and a reporter gene assay. When these experiments have been completed we will have a full molecular characterisation of the contribution of the E-boxes and effect of the conserved variant haplotype on the circadian expression of SCN5A. Our overall goal is to link circadian mechanism to these variants to explore novel treatment routes.

During early development, placental cells called trophoblasts face diverse challenges including nutrient and oxygen-limited microenvironments and changing environmental conditions. It is also during this stage that vast changes in gene expression coincide with reprogramming of cellular metabolism. However, it is unclear how trophoblasts assess and respond to their metabolic resources to achieve a coordinated effect on cell proliferation, differentiation, and placental development. Cell metabolism is conventionally viewed as a means of obtaining bioenergy for cellular homeostasis or building blocks for biomass growth. However, metabolic intermediates function as rate-limiting substrates or co-factors in a variety of epigenetic processes.

This project aims to understand the links between metabolism and epigenetic programs directing trophoblast stemness and differentiation. Key questions to be addressed are:
How does the placenta respond and adapt to changes in varying environmental conditions?
How does cellular metabolism influence epigenetic networks in placental development?
What is the impact of metabolic dysfunction on placental development and pregnancy outcome?

The candidate will utilise primary trophoblasts isolated from human term placentas as well as the recently derived human trophoblast stem cells and organoids. Additionally, training will be provided in the following state-of-the-art research techniques:
• Metabolic phenotyping and profiling using extracellular flux analysis (using the Seahorse Bioanalyzer) and metabolomics
• Gene targeting using Crispr/Cas9, si/shRNA transfection, and lentiviral transduction
• Wet-lab based approaches for RNA-seq, single-cell RNA-seq, ChIP-seq, and bisulfite-seq etc
• Bioinformatic analysis of the above sequencing data

This project will explore the unchartered field of “Placental Metabo-Devo”, i.e. the different mechanisms by which metabolism impacts on the processes governing placental development. Addressing these fundamental biological questions may help us understand novel mechanisms of placental-related pregnancy complications such as miscarriage, preeclampsia and fetal growth restriction.
Informal enquiries can be made to Dr Irving Aye: ia319@cam.ac.uk

Metformin is one of the most commonly prescribed drugs during pregnancy worldwide. It is primarily used in late pregnancy to treat gestational diabetes, but in recent years a growing number of women have been exposed to metformin during early pregnancy, particularly in the context of maternal obesity and during fertility treatment. Previous studies from our group and others suggest that pregnancies randomised to metformin treatment are less likely to develop pre-eclampsia, hence there has been growing international interest in metformin as a potential treatment or preventative drug for pre-eclampsia, which would be started early in pregnancy. However the mechanism underlying these effects remains uncertain and there is very little understanding of how metformin exposure during early pregnancy might impact on placental function and metabolism.

In late pregnancy, we have shown that metformin significantly alters placental metabolic function, in particular reducing electron transport chain activity and ATP generation. However in early pregnancy, the placenta tends to rely more heavily on nonoxidative metabolism, potentially due to high sensitivity to reactive oxygen species. In this very different metabolic milieu, it is not clear how metformin might impact on the multiple bioenergetic and biosynthetic functions of the placenta performs during the first trimester.

This project will explore the response to metformin treatment using novel trophoblast organoids as a model of first trimester placenta. We will interrogate a series of linked research questions designed to evaluate mechanisms of metformin action and inform future clinical studies on the risks and benefits of early metformin treatment. These include:
– How is metformin transported by first trimester trophoblasts?
– Does metformin treatment at clinically relevant doses alter energy production in first trimester trophoblasts?
– Does metformin treatment at clinically relevant doses alter biosynthetic function in first trimester trophoblasts?

The project will involve the opportunity to develop skills in working with both human organoid culture and trophoblast isolation. Support will be given to learn a wide range of molecular biology techniques, in addition to other key transferable skills including as data analysis and scientific writing.

Most of the defects resulting in pregnancy complications in both humans and knockout mice are associated with altered vasculature of the placenta. New emerging data suggests that the vascular endothelium (i.e. the cells that line blood vessels) play important roles as ‘signalling centres’ that contribute to growth, patterning and differentiation of surrounding tissues. We recently observed that dysregulation of the insulin-like signalling pathway in endothelial cells severely affects the growth and morphogenesis of the neighbouring syncytial epithelium layer of the mouse placenta in late gestation (PMID: 34963058). The project aims to answer the following questions: what are the signals secreted by placental endothelial cells in vivo? How do endothelial-derived signals modulate placental development and function? Do placental endothelial-derived signals also target maternal and fetal organs? The student will use a combination of single nuclei RNA sequencing and novel in vivo labelling proteomic platforms to investigate secreted protein trafficking between the endothelium and placental cell types, and determine the extent and the nature of the signals that traffic to maternal and fetal circulatory systems and organs. This project will ultimately contribute to our understanding on how the vasculature communicates with surrounding tissues and establish the potential of finding new etiological factors underlying placental pathologies, which are significant causes or fetal morbidity and mortality.

Our group is developing novel imaging methods to probe cancer metabolism in patients including deuterium metabolic imaging using oral deuterium labelled glucose, hyperpolarised MRI with intravenous carbon-13 labelled pyruvate, and Positron Emission Tomography (PET) with radiolabelled metabolites. By combining multi-omic approaches across a range of scales – from in vivo patient imaging to the cellular and subcellular scales – we are studying the molecular mechanisms driving the metabolism that we image. The tissue samples that we acquire from tumour biopsies, intraoperatively, postoperatively and following treatment, are interrogated using a range of tissue-based analytical methods such as immunohistochemistry, spatial metabolomics, and spatial transcriptomics. We are also using these techniques to understand metabolic heterogeneity and crosstalk between tumour cells and the surrounding stroma, as well as metabolic compartmentalisation within cells. By deconvolving the drivers for tumour metabolism and changes following treatment, we have shown how metabolic phenotypes can be used to better stratify tumours as aggressive, as well as for detecting early response to chemotherapy. Ultimately, the goal of the group is to use metabolism and novel imaging methods to detect cancer and response to treatment earlier in our patients. These discoveries could be used to guide potential new targets for therapy in the future.

Pancreatic cancer (PDAC) is one of the most lethal malignancies due to its late diagnosis and poor response to treatment. Development of effective therapies is impacted by a scarcity of reliable, predictive preclinical PDAC models which reflect the interactions of PDAC cells with the uniquely complex tumour microenvironment (TME). The aim of this project is to develop unique and validated experimental models that can be used to study tumour-TME-immune interactions and predict the safety and efficacy of check point inhibitor immunotherapies. We have previously shown that autologous lymphocytes can ‘reject’ PDAC organoids generated from the same patient when transplanted into immunodeficient mice in the absence of a TME. This unexpected finding could be leveraged to develop strategies to manipulate the TME in patients with PDAC to induce rejection of their own tumours. To examine the suppressive role of tumour microenvironment in modulating the response to immune system and immunotherapies, tumour-associated fibroblasts (TAFs) and macrophages (TAMs) will be isolated from the tumour samples of patients, cultured in vitro and fully characterized to define subtypes. If necessary, TAMs will also be generated by co-culture of bone marrow derived macrophages with tumour-conditioned media. TAFs and/or TAMs will be then added to the organoid-lymphocyte co-cultures in the presence or absence of immune checkpoint inhibitors. Finally, tumour organoids will be re-suspended with TAFs and/or TAMs before transplantation in vitro into humanised mice (with or devoid of macrophages) that subsequently receive checkpoint inhibitors. Non-tumour fibroblasts and macrophages will act as controls. All ethical approvals for the collection and use of human tissues, including normal and tumour tissue, as well as peripheral blood and spleen, is already in place. We have stored a range of tissues and cells, including organoids, that can be used for the proposed experiments. All animal models are also fully optimized and in routine use and will be supported by a dedicated and trained animal technician. The proposed project can thus commence immediately upon the appointment of a PhD student.

Background: IPSC-derived brain organoids are 3D in-vitro models of the developing brain particularly suited for examining early developmental events and tissue architecture (Lancaster et al., 2017; Giandomenico et al., 2019). Increased neural progenitor proliferation and excitatory/inhibitory neuron imbalance is associated with iPSC-derived 2D neurons and forebrain organoids from autistic participants with macrocephaly (Marchetto et al., 2016; Schafer et al., 2019), and in mouse models of autism (Gao et al., 2018). Studies from our group showed iPSCs from autistic individuals without macrocephaly have altered cell fates during neurodevelopment (Adhya et al., 2020). Ongoing studies have revealed that androgens increase proliferation during development in organoids. However, little is known about the effect of sex chromosomal genes on development although clinically autism shows significant bias based on biological sex. In this project we will try to understand the effect of biological sex on cell fate and proliferation during development and their interplay with sex steroid hormones. Aims and methods: We will use typical iPSCs from female and male donors to induce mutations in an autism gene associated with chromatin regulation. We will characterise developmental cell lineages from these mutant cell lines, using high-throughput single cells gene expression and chromatin regulation methods, and associated imaging methods. We will then characterise functional traits in these mutant lines. Finally, we will investigate the effect of X-chromosomal inactivation in female stem cell differentiation. This will help establish physiological chromosome X gene dosage in females. Expected outcomes: We will characterise the effect of sex differences in autism during brain development. There will be scope to undertake correlational analyses with multimodal sex differences data generated by collaborators on this project.