Supervisors and Projects
MSc by Research in Biochemistry
The following supervisors are offering MSc projects for 2024 entry:
Project Code: M1
Using novel experimental and computational approaches to understand adaptive immunity across disease contexts to defined novel therapeutic targets
We are working on deciphering how the adaptive immune system can be used as diagnostics and therapeutics. We are investigating how defects in the ability to mount effective immune responses lead to infectious disease susceptibility, impaired surveillance of cancer and immunodeficiencies, compared to the mechanisms leading to a breakdown of immunological tolerance causing autoimmune diseases. This will lead us to:
- understand why certain individuals are at greater risk of developing immunological disease
- highlight key interactions of immune cells within sites of inflammation or disease
- define novel therapeutic targets or optimal therapeutic combinations
- identify blood biomarkers immune status
- stratify patients for improved clinical management
- developing digital twins for improved clinical management
This is being achieved through the development and application of novel experimental and computational approaches, working in partnership with a global network of clinicians, immunologists and sample cohorts. We prioritise translational- and patient-focussed research to work towards bridging fundamental biology to early phase clinical trials.
How are different B cell populations developmentally linked in human health and disease?
We are investigating the generation, function and plasticity of B cell populations in human health. In particular, we are interested in how different lymphocyte subsets are developmentally linked and differences in function, and therefore providing a platform to understand how B cell fate may be different in human disease. We are defining how B cells select a particular developmental pathway, and will use this information to develop methods for modulating B cell function as potential therapeutic approaches.
How can B and T cells may be therapeutically modulated across cancers and autoimmune diseases
There is accumulating evidence for the role of both T and B cells in modulating immune responses to both solid tumours and haematological malignancies. We are investigating the contributions, function and heterogeneity of B and T cells on the immune responses to tumours and their potential role in cancer detection and treatment. We are determining the nature of B and T cell immuno-surveillance, regulation and activation across cancers and autoimmune diseases, as well as the immunological features associated with better prognosis and immunomodulation. With this, we aim to highlight novel therapeutic avenues. Our lab is affiliated with the Oxford Cancer Centre (https://www.cancer.ox.ac.uk/research/research-themes/developments-in-immuno-oncology) and non-cancer clinicians, with strong clinical links to a wide range of hard-to-treat diseases.
What is the effect of genetic and environmental variation on B and T cell fate?
Immunological health relies on a balance between the ability to mount an immune response against potential pathogens and tolerance to self. B and T cells are key to the immune response by producing antibodies and cytotoxic T cells. B/T cell clones selectively expand following antigen recognition by B and T cell receptors (BCR and TCR) respectively. BCRs are the membrane-form of antibodies and are generated through DNA recombination resulting in the potential to recognise a vast array of pathogens. Defects in the ability to mount effective B cell or T cell responses have been implicated in infectious susceptibility, impaired surveillance of cancer and immunodeficiencies, whereas a breakdown of immunological tolerance has been attributed to autoimmune diseases such as through autoantibody production and reduced numbers of regulatory B/T cells. Through integrating genomics, bulk and single-cell transcriptomics, and metabolomics data, serological, B /T cell repertoire and viromics datasets we will investigate the effect of both genetic variation and environmental factors on B cell fate, regulation, and the relationship to disease susceptibility.
Technology Development
We aim to develop novel experimental and computational tools to investigate the function of immune responses through advances in high-throughput and genetic technologies. These technologies can be readily applied to existing cohorts to investigate the immune system from unique perspectives.
Associate Prof Rachael Bashford-Rogers | Biochemistry (ox.ac.uk)
For informal enquiries: rachael.bashford-rogers@bioch.ox.ac.uk
Project Code M2
Mechanistic understanding of the ubiquitin code during cell survival and programmed cell death
Use of structural biology techniques: cryo-EM, NMR and X-ray crystallography, along with biochemical assays and biophysical techniques (including SEC-MALS, ITC and MST), to understand at a molecular level how the ubiquitin code is responsible for maintaining the balance between cell survival and cell death. Our lab has recently determined the cryo-EM structure of a large ubiquitin ligase that regulates apoptosis. Current and future research projects will follow on this and other ubiquitin ligase complexes. In addition, we investigate how binding of non-degradative ubiquitin chains can activate master kinases of inflammation, and how deubiquitinating enzymes that reverse the ubiquitin code, are regulated.
Dr Paul Elliott | Biochemistry (ox.ac.uk)
For more information about the Elliott lab see: https://elliottlab.web.ox.ac.uk
For informal enquiries: paul.elliott@bioch.ox.ac.uk
Project code M3
A novel model to study chromosome structure, biology and inheritance
At Interphase, chromosomes occupy predictable regions within the nucleus, often termed chromosome territories. Although interphase chromosomes are generally de-condensed, they form short and long-range loops in cis, interact with neighbouring chromosomes in trans, and cluster to form domains of heterochromatin or euchromatin with discrete transcriptional properties. During S-phase chromosomes replicate and cells continue to grow ahead of entering mitosis. Later, as chromosomes begin to condense in prophase, transcription declines and histone acetylation levels become reduced. Compaction continues and is maximal at metaphase, when there is a unique opportunity to physically isolate and purify individual mitotic chromosomes using flowcytometry1 and to investigate their structure and properties.
Mitotic chromosomes isolated by this approach remain biologically viable and can be induced to decondense in situ, for example when cohesion complexes are experimentally cleaved1. In addition, mitotic chromosomes that are deficient in specific chromatin components – such as DNA methylation (Dnmt1/3a/3b null), or histone H3K27me3 (PRC2 null) - are larger than their normal counterparts, while those lacking H3K9me3 appear much more compact2. Such examples provide the means to molecularly dissect mitotic chromosome structure by applying modern electron microscopy and tomography approaches3. Additionally, as mitotic chromosomes are generally devoid of factors such as MCM proteins, RNA Pol2 and transcriptional machinery1, they can be used as biological templates for reconstitution studies aimed at modelling earliest events in the transition from cytokinesis towards interphase. This is particularly important as it is not possible to purify interphase chromosomes from cells directly, and there are currently no viable alternatives or surrogates to study interphase chromosome behaviour.
Our strategy will be to purify native mitotic chromosomes and treat them with kinase inhibitors (or extracts from G1-phase cells) that induce chromosome de-compaction in vitro. Decondensed chromosomes will then be reconstituted with purified proteins that are implicated in establishing a specific interphase function. For example, using this new methodology we will ask how programs of gene expression re-established or initiated in interphase (by comparing transcription from the active and inactive X chromosomes) and how DNA is licenced for replication by the origin recognition complex (ORC), supplied with exogenous MCMs. We will employ advanced flow cytometry and microfluidic approaches to capture native mitotic chromosomes and then develop high-throughput assays to visualise and test chromosome structure. Collectively these studies will help to uncover the molecular mechanisms that underlie epigenetic inheritance as well as clarifying whether ‘modified’ mitotic chromosomes can used as a novel, experimentally tractable surrogate to model interphase.
References
1.Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction. Djeghloul D, Patel B, Kramer H, Dimond A, Whilding C, Brown K, Kohler AC, Feytout A, Veland N, Elliott J, Bharat TAM, Tarafder AK, Löwe J, Ng BL, Guo Y, Guy J, Huseyin MK, Klose RJ, Merkenschlager M, Fisher AG.Nat Commun. 2020 Aug 17;11(1):4118. doi: 10.1038/s41467-020-17823-z.PMID: 32807789
Djeghloul D, Dimond A, Cheriyamkunnel S, Kramer H, Patel B, Brown K, Montoya A, Whilding C, Wang YF, Futschik ME, Veland N, Montavon T, Jenuwein T, Merkenschlager M, Fisher AG.Nat Struct Mol Biol. 2023 Apr;30(4):489-501. doi: 10.1038/s41594-023-00943-7. Epub 2023 Mar 20.PMID: 36941433
3. Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.
Berger C, Premaraj N, Ravelli RBG, Knoops K, López-Iglesias C, Peters PJ. Nat Methods 20, 499–511 (2023). doi: 10.1038/s41592-023-01783-5. PMID: 36914814
Prof Dame Amanda Fisher | Biochemistry (ox.ac.uk)
For informal enquiries: amanda.fisher@bioc.ox.ac.uk; or lindsay.baker@bioch.ox.ac.uk
Project Code M4
Understanding and engineering microbial communities, including the human microbiome
Microbial communities contain many evolving and interacting species, which makes them difficult to understand and predict. The Foster lab combines molecular and cellular biology with ecological and evolutionary approaches to break down this complexity. Using both theory and experiment, we study how bacteria cooperate and compete in order to succeed in their communities. The lab also studies the ecological networks formed by interacting bacteria, with the goal of predicting and manipulating gut communities for better health. We have a range of projects on offer within the remit of the lab, which can be tailored to the student’s interests.
See our website for more details of our research: https://zoo-kfoster.zoo.ox.ac.uk/
Prof Kevin Foster | Biochemistry (ox.ac.uk)
For informal enquiries: Kevin.foster@biology.ox.ac.uk
Project Code M5
MSc: using mRNAs therapeutics to provide immune prophylaxis
A brief description cannot be used due to sensitive material
Associate Prof Andre Furger | Biochemistry (ox.ac.uk)
For informal enquiries: andre.furger@bioch.ox.ac.uk
Project Code M6
Control of centrosome function during the cell cycle and beyond
Background:
Centrosomes are small cytoplasmic organelles with a wide variety of cellular roles; they nucleate microtubules, serve as mitotic spindle poles, template cilia formation, while also contributing to proteostasis, signalling, trafficking and organelle positioning. They are essential for proliferation of normal cells. Indeed, congenital mutations in centrosomal genes cause growth failure syndromes (1).
Centrosomes are membrane-free organelles that concentrate up to 200 different proteins into specific structures; each centrosome contains a pair of centrioles embedded in a pericentriolar matrix. These complex multiprotein assemblies undergo dynamic changes during the cell cycle (2, 3). For instance, a single new centriole is built on each old centriole during S phase, whereas the pericentriolar matrix, the main site of microtubule nucleation, undergoes expansion and remodelling before mitosis to boost microtubule production for spindle formation.
Phosphorylation-dependent regulation of centrosome components is likely to be vital for a range of centrosome functions, including microtubule nucleation. Although several kinases including Aurora-A, Nek2, Cdk1 and Polo-like kinases 1, 2 and 4, have been implicated in centrosome regulation, a comprehensive list of their targets in the organelle is still lacking. We are currently undertaking an unbiased proteomic survey to elucidate centrosomal targets for these kinases.
Aims:
The ultimate goal of this DPhil project will be to improve our understanding of how centrosome function (i.e. biogenesis, homeostasis and microtubule nucleation) is regulated during the cell cycle and in quiescence. First, we will functionally characterise newly identified phosphorylation sites in centrosomal proteins with an established role in these processes. By developing tools such as specific antibodies against phosphorylation sites along with cell lines where the sites are genetically edited, we will obtain mechanistic insight into how these post-translational modifications contribute to centrosome function. Second, we will ask if these phosphorylation sites are present across multiple cell types, and if so, whether their impact on centrosome function is the same. Results from our laboratory suggest that erythroid progenitors do not remodel their centrosomes during mitosis and therefore certain aspects of centrosome function are regulated in a cell type-specific manner (4). In these instances, post-translational modifications of centrosome components could play an even more important part. This hypothesis will be tested with the tools generated in the first aim, and proteomic survey of centrosomes from these unusual cell lineages will provide further insight. Finally, armed with knowledge from the first two aims, we will ask whether and how centrosomal microtubule nucleation capacity impacts on cellular processes such as trafficking, signalling and spindle formation.
Visit us on https://gergelylab.com
(1) Chavali, Putz and Gergely. 2014. The role of centrosomes in development and disease. Philos Trans R Soc Lond B Biol Sci.
(2) Tischer, Carden and Gergely. 2021. Accessorizing the centrosome: new insights into centriolar appendages and satellites. Current Opinion in Structural Biology.
(3) Vasquez-Limeta and Loncarek. 2021. Human centrosome organization and function in interphase and mitosis. Seminars in Cell & Developmental Biology.
(4) Tatrai and Gergely. 2022. Centrosome function is critical during terminal erythroid differentiation. The EMBO Journal.
For more information about the Gergely lab see: https://gergelylab.com
Dr Fanni Gergely | Biochemistry (ox.ac.uk)
For informal enquiries: fanni.gergely@bioch.ox.ac.uk
Project Code M7
Regulation of organelle homeostasis and dynamics by membrane contact sites
Brief description of project(s) or research theme (no more than 500 words): The eukaryotic cell harbour a set of membrane-bound organelle. Coordination of the activity within all of them is dependent on membrane contact sites. These contact sites serve as platform to exchange metabolites (such as lipids) and information between compartments. We study how membrane contact sites allow organelle biogenesis by providing the appropriate complement of lipid molecule for the build up of their membranes. We assess the effect of impairing these contact sites on organelle growth, functions and dynamics. To achieve these goals, we use protein structure prediction, high-end microscopy, functional genomics and mass-spectrometry-based lipidomics. Research projects are available in various directions. For instance, our recent discovery of a new structural interaction module between Miro, a key mitochondrial protein and a factor in Parkinson’s disease, and several interactors found often on different organelles paves the way to decipher the physiological role of this interaction, which will be tested by CRISPR-mediated genome editing. Another direction is about the effect of membrane lipid composition and membrane transporter activity. Membrane contact sites are sites of lipid transport and their dysfunction may cause aberrant membrane composition. Because membrane lipids are the solvent in which membrane transporter operates, it is expected that membrane composition should influence transporter activity. Indeed, through e genomics screen, we found indeed the first evidence that this could be the case. Interestingly, improper lipid transport between organelles led to decreased activity of plasma membrane transporters, which subsequently underwent endocytosis. A project is thus to study how membrane composition affects transporter activity, and what signal triggers their internalization. Several other projects are available and can be discussed with the candidates.
For more information about the Kornmann lab see: http://www.kornmann.group
Prof Benoit Kornmann | Biochemistry (ox.ac.uk)
For informal enquiries: benoit.kornmann@bioch.ox.ac.uk
Project Code M8
Maintenance of genome integrity through DNA repair
Preserving genome integrity through DNA repair is critical for human health and defects in these pathways leads to a variety of pathologies including neurodegeneration and cancer. Our research aims to understand the mechanistic basis of DNA repair, with specific reference to how a family of proteins called Poly(ADP-ribose) polymerases (PARPs) regulate these processes. Our long-term vision is to exploit this knowledge to treat a variety of diseases including neurodegeneration and cancer.
Research Projects
PARPs are a cornerstone of the DNA damage response that promote repair of breaks in the DNA helix by modifying proteins at the damage site with ADP-ribose - a process known as ADP-ribosylation. These pathways are critical to maintain genome integrity and are attractive clinical targets, with PARP inhibitors being used to treat breast and ovarian tumours. However, despite the success of PARP inhibitors in the clinic, the mechanistic basis of how PARPs regulate DNA repair is unclear, representing a fundamental barrier to refine and broaden the application of these agents in the clinic. The overall goal of our research is to address these fundamentally important questions by defining the mechanistic basis of how PARPs regulate a variety of DNA repair mechanisms. DPhil projects are available that integrate cutting edge genome engineering, proteomics and cell biology to address the following:
- How do PARPs become activated in response to DNA damage?
- How do these modifications regulate DNA repair?
- What regions of the genome are hotspots for PARP-dependent DNA repair.
Prof Nick Lakin | Biochemistry (ox.ac.uk)
For informal enquiries: Nicholas.lakin@bioch.ox.ac.uk
Project Code: M9
Cytoskeletal organisation of the malaria parasite
The malaria parasite, Plasmodium falciparum, infects various cell types within its insect and human hosts as part of a complex life cycle (1). In the human bloodstream, the parasite invades red blood cells to replicate inside, hidden away from the host immune system. To invade these cells, the parasite uses specialised invasive organelles, the rhoptries and micronemes (2).
The parasite cell needs to be precisely organised to successfully invade the host. The rhoptries and micronemes are held at the apical end of the cell, whereas its nucleus, mitochondria and other organelles are held toward the basal end (3). It is not known how the cell sets up and maintains this striking polarisation. One class of proteins proposed to contribute are the various cytoskeletal filaments that form fascinating structures throughout the parasite. This is exemplified by the microtubules, which form a beautiful array along the parasite membrane (4). However how these filaments link to other structures in the cell to carry out their function remains mysterious. Our lab is interested in how these filament systems position and shape organelles throughout the parasite cell. Our primary techniques are in vitro structural biology and biophysics, though we are expanding into in vivo techniques to examine these proteins within the malaria parasite itself.
The aim of this DPhil project is to further our understanding into how organelles are linked to the microtubule cytoskeleton in the malaria parasite. We will explore the following questions: We will identify and characterise novel microtubule-binding proteins to see how they link organelles to microtubules in vitro and in vivo. We will examine how they recruit organelles to the cytoskeleton using electron microscopy, then design assays to test their functional dynamics using biophysical techniques including TIRF microscopy. To dissect the protein function in cells we will use CRISPR/Cas9-based strategies to modify the protein inside the parasite. We will explore their localisation in cells and examine the consequences of knocking out the protein using light microscopy.
Please contact clinton.lau@bioch.ox.ac.uk,
For more information about the Lau lab see: https://laulab.web.ox.ac.uk/.
References
- Venugopal, K. et al. Nat. Rev. Microbiol. 2020 183 18, 177–189 (2020). https://doi.org/10.1038/s41579-019-0306-2
- Schrevel, J. et al. Parasitology 135, 1–12 (2008). https://doi.org/10.1017/S0031182007003629
- Fowler, R. E. et al. Parasitology 117, 425–433 (1998). https://doi.org/10.1017/S003118209800328X
- Ferreira, J. et al. bioRxiv (2022). https://doi.org/10.1101/2022.04.13.488170
Dr Clinton Lau | Biochemistry (ox.ac.uk)
For informal enquiries from prospective students: Clinton.lau@bioch.ox.ac.uk
Project Code M10
Toll-mediated regulation of intestinal stem cells
Toll-like receptors (TLRs) regulate the crosstalk between innate and adaptive immunity. They interact with evolutionary conserved pathogen-derived molecules but also with host ligands (damage/danger-associated molecules) that derive from injured tissues. This is probably why TLRs have been found to be expressed on several kinds of stem/progenitor cells including intestinal stem cells (ISCs). In some of these cells, the role of TLRs in the regulation of basal motility, proliferation, differentiation, self-renewal, and immunomodulation of SCs has been demonstrated.
However, the regulatory mechanisms by which TLRs may control ISC proliferation in normal conditions and in response to injury or infection are still unexplored. This is where the use of the vinegar fly Drosophila melanogaster can lead to faster progress. TLRs have been named after the identification of the TOLL gene in Drosophila more than 30 years ago, and the signalling pathway downstream of TOLL has been shown to be evolutionary conserved in innate immunity of flies, mice, and humans. Our published and unpublished work (the latter leading to the current proposal) has shown that the classical TOLL pathway is involved in the maintenance of gut bacteriome and in ISC renewal following infection. Our proposal is to identify the mechanisms by which TOLL is regulating ISCs and how downstream of TOLL, NF-κB and JNK orchestrate self-renewal and tissue regeneration.
Using the rich “toolbox” of Drosophila we will identify how inducing Toll activity in ISCs triggers cell division and what are the downstream targets of the Toll/NF-κB pathway. We know that one of these is JNK a signal that maintains “stemness” in the Drosophila gut. By conditional activation of the Toll ligand, Spaetzle (Spz), we will investigate whether it acts as a mitogen. Triggering Toll in ISCs activates Toll targets in other progenitor cell types as well as enterocytes and so either Spz or another cytokine-like molecule may act as a mitogen across the intestinal epithelium under stress or infection.
Prof Petros Ligoxygakis | Biochemistry
For informal enquiries: petros.ligoxygakis@bioch.ox.ac.uk
Project Code M11
What drives the evolution of epigenetic mechanisms?
Epigenetic regulation is fundamental to development as it establishes heritable changes in gene expression without affecting the underlying DNA sequence. Many epigenetic mechanisms exist, which specify gene expression states and have the potential to be transmitted through cell division. These epigenetic mechanisms are often widely conserved across eukaryotes. However, despite their ancient origin, many epigenetic mechanisms have been lost independently numerous times in different eukaryotic lineages. Why this happens is very puzzling. Recently we discovered a potential explanation for why one epigenetic pathway, cytosine DNA methylation (5-methyl-Cytosine), conserved from plants to humans, is frequently lost in different eukaryotes. We discovered that cytosine DNA methyltransferases can damage the DNA, introducing a form of alkylation damage, 3-methyl-cytosine, which is highly toxic (Rosic et al., Nature Genetics, 2018). This toxic effect results in the co-evolution of a DNA repair pathway, ALKB2, which is specialised to remove 3-methyl-cytosine from DNA. ALKB2 is present in species that have DNA methylation but absent from those that have lost the methylation machinery. Loss of ALKB2 may therefore predispose organisms to lose DNA methylation. This work acts as an example of how, more generally, epigenetic mechanisms might be lost due to negative consequences associated with them. In this PhD project we aim to identify other potential examples of costs associated with epigenetic mechanisms. To do this we will use co-evolution across species, using computational methods, to identify genes that are associated with particular epigenetic mechanisms across evolution. Importantly, we will then explore whether similar relationships are found in cancer, because epigenetic mechanisms are frequently lost or inactivated in cancer cells. The project will involve computational analyses of large datasets, in particular comparative genomics across eukaryotic species and gene expression analyses across multiple cancers, including single cell sequencing data analysis. The work will identify new explanations for why epigenetic pathways evolve, and link these to factors driving the evolution of cancer cells and the progression of the disease.
To find out more about the Sarkies lab see: https://psarkies.wixsite.com/epievo
Associate Prof Peter Sarkies | Biochemistry (ox.ac.uk)
For informal enquiries: Peter.sarkies@bioch.ox.ac.uk
Project Code M12
Interrogating nuclear structure-function relationships in mammalian cells by advanced super-resolution imaging
Three-dimensional (3D) chromatin organisation plays a crucial role in regulating mammalian genome functions such as RNA transcription, replication and DNA repair. Population-based sequencing approaches (e.g. Hi-C) have highlighted the compartmentalisation of chromatin into 0.5-1 MB sized topologically associating domains (TADs). However, many of the physical features at the single-cell level are still underexplored. Our primary research objective is to identify principles and underlying mechanisms of functional chromatin organisation in mammalian cells. Specifically, we aim to understand the interplay between biophysical forces, epigenetic memory, and cohesin complex activity to modulate cell-type-specific transcriptional programmes by directly visualising dynamic nuclear organisation and gene activity in living or 3D-preserved cells. To this end, we employ a combination of genetic editing with innovative in vivo/in situ fluorescence labelling and super-resolution imaging approaches. Our activities are closely linked to the Micron Oxford Advanced Bioimaging Unit and supported by our well-established ties to leading chromatin and epigenetic research groups within the Department and across Oxford.
For a MSc/PhD project, we seek (an) enthusiastic, proactive and adventurous student(s) eager to immerse in the latest imaging technologies to study topographical and biophysical aspects of gene regulation in an interdisciplinary environment. The topic of the project can be along the lines of either (1) studying single nucleosome dynamics within mesoscale chromatin domains using correlative single molecule tracking and super-resolution SIM imaging, (2) analysing loop-extruding and sister chromatid cohesive and loop-extruding cohesin complexes by super-resolution expansion microscopy (ExM) and/or super-resolution 3D correlative light and electron microscopy (CLEM), (3) studying the effect of directed phase separation on mesoscale domain organisation and transcriptional modulation, (4) examining mechanisms of gene reactivation during pathological de-differentiation processes (e.g. liver fibrosis, EMT), or (5) examining enhancer-promoter interactions e.g. in the alpha-globin locus, using multiplexed RNA-DNA-Immuno-FISH and correlative 3D super-resolution light end electron microscopy. The details of any project will be subject to personal preferences and be worked out closer to start date.
Main techniques: Mammalian tissue culture, molecular cloning, transfection, immunofluorescence labelling, fluorescence in situ hybridisation (DNA/RNA FISH), super-resolution structured illumination microscopy, single-molecule imaging, focussed ion beam scanning electron microscopy (FIB-SEM), computational image analysis.
Relevant papers:
Miron E, ..., Schermelleh L. 2020. Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin. Science Advances 6, eaba8811.
Brown JM, … Schermelleh L, Buckle VJ. 2022. RASER-FISH, a non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protoc 17, 1306-1331.
Rodermund L,..., Schermelleh L#, Brockdorff N#. 2021. Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading. Science. 372: eabe7500
Ochs F, ..., Schermelleh L#, Lukas J#, Lukas C. 2019. Stabilization of chromatin topology safeguards genome integrity. Nature, 594: 571-574.
Schermelleh L et al. 2019. Super-resolution microscopy demystified. Nat Cell Biol 21: 72-84.
Associate Prof Lothar Schermelleh | Biochemistry (ox.ac.uk)
For informal enquiries: lothar.schermelleh@bioch.ox.ac.uk
Project Code M13
Protein structure and interactions in health and disease.
Our laboratory seeks to understand how protein functions arise from their molecular structure, conformational changes, and interactions, and how these processes are involved in human health and disease. Projects in at least three areas are possible: (1) We have a long-standing interest in the molecular mechanisms by which Influenza virus proteins function. To this end, we are studying the protein-protein and lipid interactions of ‘flu’ proteins to better understand their role in the virus life cycle and to identify potential therapeutic targets. (2) A collaborative project to combine experimental and computational tools to determine the timescale and energetics of conformational changes in proteins. (3) Investigations into the molecular mechanisms underlying chaperone protein activity, to explain how they interact with misfolded/unfolded clients.A central technique of our laboratory is solution nuclear magnetic resonance (NMR) spectroscopy, which allows atomic-level studies of protein structures and their interactions. NMR can be uniquely informative in situations where the molecular conformations or interactions are dynamic or heterogeneous. However, we also use a wide variety of other biochemical and biophysical tools as needed for these investigations. We have collaborations with various research groups including virologists, cell biologists, and computational biologists.
Associate Prof Jason Schnell | Biochemistry (ox.ac.uk)
For informal enquiries: Jason.Schnell@bioch.ox.ac.uk
Project Code M14
The molecular mechanism of establishment of sister chromatid cohesion at a single molecule resolution.
The error-free duplication and segregation of chromosomal DNAs is fundamental to cell proliferation. Sister DNAs generated during DNA replication must be held together from S phase until anaphase, when they finally disjoin to opposite poles of the cell. This process called sister chromatid cohesion (cohesion) ensures equal segregation of the genome. Cohesion results from entrapment of sister-DNAs within a highly conserved, ring-shaped protein complex called cohesin, with proteolytic cleavage of the cohesin rings being initiated prior to cell division to enable disjunction of sister chromatids. Cohesin is essential for orderly segregation of chromosomes during cell division, and when its function is perturbed, this can lead to human cancers and developmental disorders. However, despite the fundamental importance of cohesion for cell division, the mechanisms that control how cohesion is created remain enigmatic and constitute a major gap in our understanding of eukaryotic biology. Our research goal is to understand the molecular mechanism of sister chromatid cohesion. We have recently demonstrated that cohesin rings that trap unreplicated DNAs in G1 are ‘converted’ into cohesive molecules (trapping both sister DNAs) during replication (doi: 10.7554/eLife.56611). One can envisage two scenarios for how DNA associated cohesin rings become cohesive. 1. The replication machinery could simply pass through the large cohesin rings, resulting in entrapment of the replicated DNAs inside the rings. 2. Cohesin rings could be removed from ahead of the fork and deposited behind the fork (like the nucleosomes are). In order to test these two scenarios, we have recently set up a single molecule assay to track the fate of cohesin rings when they encounter the replication machinery on DNA (https://doi.org/10.1101/2022.09.15.508094) . This project will build upon this single molecule assay to test the requirement of the opening of cohesin rings 3 interfaces in order to establish cohesion. This will involve biochemical techniques like purification of human/Xenopus cohesin complex expressed in insect cells, fluorescent labelling of the purified complex and chemical crosslinking of any one or all 3 cohesin ring interfaces. This will be followed by single molecule imaging of replisome and cohesin. The in vitro single molecule assays will be complemented by in vivo analysis of single molecules of cohesin, we have recently begun single particle tracking of cohesin molecules inside living cells. The goal would be to observe cohesion establishment in vivo at a single molecule level. The in vivo work will involve CRISPR/Cas9 editing of human and DT40 cells to enable fluorescent labelling of cohesin complex and replisome components. The imaging will be done with a custom built TIR microscope as well as the ONI nano imager. We are a small, friendly, inclusive, supportive, and highly focussed group. As a Dphil/ MSc (res) student you will be very well looked after. Don’t hesitate to contact me if you need further information or a tour of the lab.
Dr Madhusudhan Srinivasan | Biochemistry (ox.ac.uk)
For informal enquiries madhusudhan.srinivasan@bioch.ox.ac.uk
Project Code M16
Replication barriers and genome stability
A hallmark of ageing is the accumulation of genomic mutations and rearrangements through mistakes made during the normal processes of DNA replication, repair and chromosome segregation. It is thought that this gradual corruption of the genome results in gene regulatory changes, which cause cellular degeneration and functional decline that ultimately drives ageing and its associated diseases. Accordingly, the pace of genomic deterioration is likely to be a key determinant of healthy lifespan, which is strongly influenced by both environmental and genetic factors. Through a complete understanding of how mutations and genome rearrangements arise, as well as the factors that mitigate their occurrence, we will be better placed to develop new approaches to improve the healthy ageing of humankind.
Conflicts between replication forks and single-strand DNA breaks (SSBs) and protein-DNA complexes (PDCs) are a major threat to genome stability through their potential to cause fork collapse and failure of complete genome duplication. By exploiting state-of-the-art fission yeast genetics, advanced microscopy, protein biochemistry, advanced proteomics and genomic approaches, we aim to elucidate the different pathways that limit genome instability arising from replication fork- SSB/PDC conflicts, and how pathway choice is influenced by the nature and context of the SSB/PDC. This work will make a seminal contribution to our understanding of how genome deterioration, and consequent ageing and age-related disorders, is driven by problems that arise during S phase.
For more information about the Whitby lab see: https://whitbylab.com
Prof Matthew Whitby | Biochemistry (ox.ac.uk)
For informal enquiries: matthew.whitby@bioch.ox.ac.uk
DPhil in Biochemistry
The following supervisors are offering DPhil projects for 2024 entry:
Project code M1
Understanding death in bacterial communities using electron cryotomography
Programmed cell death (PCD) is a genetically regulated process of cell death. It has been well studied in multicellular organisms, where it is critical in development, infection, and cancer. However, this fundamental process has largely been overlooked in bacteria. Recent research has shown that bacterial communities can induce PCD in a portion of their population. Critically, it has been demonstrated that programmed death pathways in bacteria can be triggered by antibiotics, starvation and viral infection, although the mechanisms of death remain unclear. The activation of programmed cell death in bacteria by antibiotics has significant implications for antibiotic resistance, infection and immune responses, and commensal microbiomes in humans. Although much research has focused on the mechanisms by which bacteria evade death in response to antibiotics, very little is known about what happens to the cells that do die. To deduce new ways of killing bacteria, it is important to understand the fundamental biology underlying their death.
While the morphological changes in mammalian cells during apoptosis are easily observed by light microscopy, higher resolution imaging methods such as electron cryomicroscopy (cryoEM) are required for observing cellular ultrastructure changes in bacteria, which are much smaller. Electron cryotomography (cryoET), a cryoEM modality where a series of images at different angles to the electron beam is used to reconstruct 3D maps of pleiomorphic objects such as cells, in particular can provide high resolution structural and contextual information about bacteria. Advanced equipment for cryoEM, for thinning biological samples with a focused ion beam (FIB), and a new cryo-fluorescence microscopy facility are all available to conduct these experiments within the Department.
For informal enquiries: lindsay.baker@bioch.ox.ac.uk
Dr Lindsay Baker | Biochemistry (ox.ac.uk)
Project Code: D2
Using novel experimental and computational approaches to understand adaptive immunity across disease contexts to defined novel therapeutic targets
We are working on deciphering how the adaptive immune system can be used as diagnostics and therapeutics. We are investigating how defects in the ability to mount effective immune responses lead to infectious disease susceptibility, impaired surveillance of cancer and immunodeficiencies, compared to the mechanisms leading to a breakdown of immunological tolerance causing autoimmune diseases. This will lead us to:
- understand why certain individuals are at greater risk of developing immunological disease
- highlight key interactions of immune cells within sites of inflammation or disease
- define novel therapeutic targets or optimal therapeutic combinations
- identify blood biomarkers immune status
- stratify patients for improved clinical management
- developing digital twins for improved clinical management
This is being achieved through the development and application of novel experimental and computational approaches, working in partnership with a global network of clinicians, immunologists and sample cohorts. We prioritise translational- and patient-focussed research to work towards bridging fundamental biology to early phase clinical trials.
How are different B cell populations developmentally linked in human health and disease?
We are investigating the generation, function and plasticity of B cell populations in human health. In particular, we are interested in how different lymphocyte subsets are developmentally linked and differences in function, and therefore providing a platform to understand how B cell fate may be different in human disease. We are defining how B cells select a particular developmental pathway, and will use this information to develop methods for modulating B cell function as potential therapeutic approaches.
How can B and T cells may be therapeutically modulated across cancers and autoimmune diseases
There is accumulating evidence for the role of both T and B cells in modulating immune responses to both solid tumours and haematological malignancies. We are investigating the contributions, function and heterogeneity of B and T cells on the immune responses to tumours and their potential role in cancer detection and treatment. We are determining the nature of B and T cell immuno-surveillance, regulation and activation across cancers and autoimmune diseases, as well as the immunological features associated with better prognosis and immunomodulation. With this, we aim to highlight novel therapeutic avenues. Our lab is affiliated with the Oxford Cancer Centre (https://www.cancer.ox.ac.uk/research/research-themes/developments-in-immuno-oncology) and non-cancer clinicians, with strong clinical links to a wide range of hard-to-treat diseases.
What is the effect of genetic and environmental variation on B and T cell fate?
Immunological health relies on a balance between the ability to mount an immune response against potential pathogens and tolerance to self. B and T cells are key to the immune response by producing antibodies and cytotoxic T cells. B/T cell clones selectively expand following antigen recognition by B and T cell receptors (BCR and TCR) respectively. BCRs are the membrane-form of antibodies and are generated through DNA recombination resulting in the potential to recognise a vast array of pathogens. Defects in the ability to mount effective B cell or T cell responses have been implicated in infectious susceptibility, impaired surveillance of cancer and immunodeficiencies, whereas a breakdown of immunological tolerance has been attributed to autoimmune diseases such as through autoantibody production and reduced numbers of regulatory B/T cells. Through integrating genomics, bulk and single-cell transcriptomics, and metabolomics data, serological, B /T cell repertoire and viromics datasets we will investigate the effect of both genetic variation and environmental factors on B cell fate, regulation, and the relationship to disease susceptibility.
Technology Development
We aim to develop novel experimental and computational tools to investigate the function of immune responses through advances in high-throughput and genetic technologies. These technologies can be readily applied to existing cohorts to investigate the immune system from unique perspectives.
Associate Prof Rachael Bashford-Rogers | Biochemistry (ox.ac.uk)
For informal enquiries: rachael.bashford-rogers@bioch.ox.ac.uk
Project Code D3
Nanomachines in the bacterial cell envelope
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The cell envelope of bacteria comprises the cell wall and either one or two membranes, and provides a formidable barrier to the movement of macromolecules between the bacterial cytoplasm and the external environment. Our group aims to understand the molecular mechanisms by which proteins, nucleic acids, and mechanical force are transferred across and along these barriers. As part of this work we characterise the dedicated nanomachines that carry out these processes.
We use a wide range of methodologies in our work, in some cases via collaboration. These approaches include protein purification and characterisation, bacterial cell biology, bacterial genetics, live cell single molecule fluorescence imaging and other biophysical analysis, bioinformatics, and structural biology.
Specific research areas in which projects could be offered include:
- Protein transport. We study the transport of folded proteins across the bacterial inner membrane by the Tat transport system and protein export across the outer membrane by the recently discovered Type 9 Secretion System. Both systems are important for bacterial pathogenesis. We are also interested in the export of lipoproteins to the surface of Gram-negative bacteria.
- DNA transport. We study the mechanisms by which genes move between bacteria, thereby contributing to antibiotic resistance and other adaptive traits. We are interested in the processes of Conjugation, in which DNA is transferred between bacteria either by direct contact or via a retractile pilus, and Transformation (natural competence) in which the bacterium takes up naked DNA molecules directly from their environment.
- Gliding motility in which bacteria move rapidly across solid surfaces using surface adhesins running on mobile tracks located in the cell envelope.
- Physical properties of the cell envelope. In particular, we are interested in the functional properties of the periplasm, which is the compartment lying between the inner and outer membrane of Gram-negative bacteria and which contains the cell wall.
More details about my group can be found at: https://benberksgroup.web.ox.ac.uk
Example references showcasing some of our technical approaches:
- Hennell James et al. (2021) Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility. Nat Microbiol 6: 221–233.
- Lauber et al. (2018) Type 9 secretion system structures reveal a new protein transport mechanism. Nature 564: 77–82.
- Alcock et al. (2016) Assembling the Tat protein translocase. Elife 5: e20718.
- Alcock et al. (2013) Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system. PNAS 110: E3650–E3659.
- Silale et al. (2021) The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation. Mol Microbiol 116: 416-426.
Prof Ben Berks | Biochemistry (ox.ac.uk)
For more information about the Berks lab see: https://benberksgroup.web.ox.ac.uk
For informal enquiries: ben.berks@bioch.ox.ac.uk
Project Code D4
TPC2 Channels – Selectivity, gating and drug modulation
Two-pore channels (TPCs) are ubiquitous cation channels expressed on acidic organelles. TPC2 localizes to lysosomes where it mediates numerous patho-physiologically-relevant roles from the trafficking of coronaviruses and growth factors through to triggering global calcium signals. It is highly unusual given its ability to switch its ion selectivity in response to the physiological activators PI(3,5)P2 and NAADP. In essence, it toggles between a sodium-selective and non-selective (calcium-permeable) cation channel. But the relationship between these distinct modes of action is currently unclear.
Recent work has identified i) small molecule agonists that mimic the distinct effects of PI(3,5)P2 and NAADP on TPC2, and ii) NAADP-binding proteins (the elusive 'receptors') that associate with TPCs to mediate indirect NAADP activation.
This studentship will explore these aspects using molecular simulation methodologies in conjunction with collaborators based at UCL and Cambridge. To explore the underlying mechanisms coupling gating and ion selection, we will employ molecular dynamics simulations to predict conformational states associated with permeation and ligand binding which in turn will be validated by comparing the functional properties of select TPC2 mutants (by our collaborators). We will also use molecular dynamics to identify the molecular determinants of NAADP receptor-TPC2 interactions. The studentship will build on current work being performed in the lab as part of the wide collaboration.
The student will become proficient in MD, docking and other simulation methodologies as well as python.
Prof Phil Biggin | Biochemistry (ox.ac.uk)
For informal enquiries: Philip.biggin@bioch.ox.ac.uk
Project Code D5
Analysis of the human antibody response in volunteers vaccinated with novel vaccines targeting the essential RH5 invasion complex used by P. falciparum malaria parasites to invade red blood cells.
Traditionally the analysis of human antibody responses following vaccination has involved global approaches, with assays measuring the total polyclonal serum antibody response in terms of titre, concentration, subtype response or avidity. Although such measures remain useful readouts, the development of highly effective vaccines against difficult and complex pathogens, such as the Plasmodium parasite that causes human malaria, requires a much greater understanding of the fine specificity of the vaccine-induced antibody response. In recent years, significant advances have been made in terms of ability to analyse the human antibody repertoire generated in response to vaccination, and to assess the identification of key functional epitopes.
The aim of this project will be to analyse the human antibody response induced by novel candidate vaccines targeting the RH5-invasion blood-stage malaria invasion complex in Phase 1/2 clinical trials undertaken in Oxford and East or West Africa. This will build upon on-going research in the Draper Group to develop vaccines against this critical target complex within the parasite and to better understand its biology and the critical antibody functions that inhibit parasite growth. A variety of techniques will be used to isolate antigen-specific B cell subsets from volunteers immunised with the RH5-complex antigens using different vaccine delivery regimens in humans (adults and infants). The isolated B cells will be used to analyse the human antibody response via sequencing and cloning of the B cell receptor gene repertoire. Key analyses will include variable region gene usage, mutation, and clonality of the response over time post-immunisation. Identified sequences will be used to generate human monoclonal antibodies (hu-mAbs) which will be assessed for functional anti-parasitic activity, affinity and epitopes mapped on the antigens using structural, biophysical and immunological approaches. The outputs of this work should identify key epitopes around this protein complex and determinants within it that are recognised following human vaccination with these novel vaccines. Next steps could include rational design of improved next-generation vaccine immunogens for onward clinical development, or immunological experiments to understand how antibodies mediate anti-parasitic function. The project will be benefit from the group’s extensive experience of clinical immunology, B cell immuno-monitoring, protein engineering and parasitology, as well as from strong collaborations with other leading labs globally.
For more information on the Draper Lab, please see: https://draperlab.web.ox.ac.uk/
Prof Simon Draper | Biochemistry (ox.ac.uk)
For informal enquiries: simon.draper@bioch.ox.ac.uk
Project Code D6
Mechanistic understanding of the ubiquitin code during cell survival and programmed cell death
Use of structural biology techniques: cryo-EM, NMR and X-ray crystallography, along with biochemical assays and biophysical techniques (including SEC-MALS, ITC and MST), to understand at a molecular level how the ubiquitin code is responsible for maintaining the balance between cell survival and cell death. Our lab has recently determined the cryo-EM structure of a large ubiquitin ligase that regulates apoptosis. Current and future research projects will follow on this and other ubiquitin ligase complexes. In addition, we investigate how binding of non-degradative ubiquitin chains can activate master kinases of inflammation, and how deubiquitinating enzymes that reverse the ubiquitin code, are regulated.
Dr Paul Elliott | Biochemistry (ox.ac.uk)
For more information about the Elliott lab see: https://elliottlab.web.ox.ac.uk
For informal enquiries: paul.elliott@bioch.ox.ac.uk
Project Code D7
Molecular and structural mechanisms for accurate 3’-splice site selection during mRNA synthesis by the human spliceosome
Mammalian genes are transcribed into precursor messenger RNAs (pre-mRNAs), from which the spliceosome excises non-coding introns in the nucleus before the mRNA is exported to the cytoplasm and translated into proteins.
The spliceosome is a dynamic RNA-protein complex that assembles the novo on each pre-mRNA and catalyses two sequential reactions – branching and exon ligation – to produce mRNAs with continuous protein-coding information. Following branching, the ATPase Prp16 remodels the spliceosome to promotes docking of the 3’ splice site (3’-SS) in the active site, where it is stabilised by specific exon ligation factors. After exon ligation, the ATPase Prp22 releases the mature mRNA for export to the cytoplasm. Importantly, Prp22 also proofreads docking of the 3’-SS to ensure correct mRNA synthesis
Electron cryo-microscopy (cryo-EM) has revealed the molecular structures of the branching and exon ligation conformations of the spliceosome, showing how splice sites are recognised and how specific factors stabilize each catalytic conformation
Recently, using an in vitro yeast system, we have uncovered additional intermediates during Prp16 remodelling, suggesting there are unexplored dynamics during selection and docking of 3’-SS in the active site
These novel exon ligation factors may bind the spliceosome sequentially and act as chaperones to modulate fidelity of 3’-SS selection by forming short-lived, transient spliceosome intermediates. To understand how these factors modulate spliceosome dynamics and to capture transient intermediates, we aim to develop an in vitro system for the catalytic stage of splicing and study spliceosome remodelling during catalysis using time-resolved cryo-EM. We aim to establish a vitrification set-up that combines time control of the reaction with spray-based grid deposition (currently under development at eBIC) and assessment of complex composition using mass photometry, for which we are developing a collaboration with Philipp Kukura’s group in Chemistry. As a dynamic complex remodelled by ATP, the spliceosome offers an ideal sample for the development of time-resolved cryo-EM methods to access transient states that may be in thermodynamic equilibrium and thus inaccessible to conventional cryo-EM sample preparation methods. This work would unravel the molecular mechanisms by which several disease-associated splicing factors modulate correct mRNA synthesis and alternative splicing during splicing catalysis.
References
1. Wilkinson, M. E., Charenton, C. & Nagai, K. RNA Splicing by the Spliceosome. Annual Review of Biochemistry 89, 1–30 (2019).
2. Fica, S. M., Oubridge, C., Wilkinson, M. E., Newman, A. J. & Nagai, K. A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation. Science (New York, NY) 363, 710–714 (2019).
3. Dybkov, O. et al. Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins. Sci Adv 9, eadf1785 (2023).
4. Wilkinson, M. E., Fica, S. M., Galej, W. P. & Nagai, K. Structural basis for conformational equilibrium of the catalytic spliceosome. Mol Cell (2021) doi:10.1016/j.molcel.2021.02.021.
For more information about the Fica lab see: https://snrnpsplicingbiochemlab.web.ox.ac.uk/
Dr Sebastian Fica | Biochemistry (ox.ac.uk)
For informal enquiries: Sebastian.fica@bioch.ox.ac.uk
Project Code D8
A novel model to study chromosome structure, biology and inheritance
At Interphase, chromosomes occupy predictable regions within the nucleus, often termed chromosome territories. Although interphase chromosomes are generally de-condensed, they form short and long-range loops in cis, interact with neighbouring chromosomes in trans, and cluster to form domains of heterochromatin or euchromatin with discrete transcriptional properties. During S-phase chromosomes replicate and cells continue to grow ahead of entering mitosis. Later, as chromosomes begin to condense in prophase, transcription declines and histone acetylation levels become reduced. Compaction continues and is maximal at metaphase, when there is a unique opportunity to physically isolate and purify individual mitotic chromosomes using flowcytometry1 and to investigate their structure and properties.
Mitotic chromosomes isolated by this approach remain biologically viable and can be induced to decondense in situ, for example when cohesion complexes are experimentally cleaved1. In addition, mitotic chromosomes that are deficient in specific chromatin components – such as DNA methylation (Dnmt1/3a/3b null), or histone H3K27me3 (PRC2 null) - are larger than their normal counterparts, while those lacking H3K9me3 appear much more compact2. Such examples provide the means to molecularly dissect mitotic chromosome structure by applying modern electron microscopy and tomography approaches3. Additionally, as mitotic chromosomes are generally devoid of factors such as MCM proteins, RNA Pol2 and transcriptional machinery1, they can be used as biological templates for reconstitution studies aimed at modelling earliest events in the transition from cytokinesis towards interphase. This is particularly important as it is not possible to purify interphase chromosomes from cells directly, and there are currently no viable alternatives or surrogates to study interphase chromosome behaviour.
Our strategy will be to purify native mitotic chromosomes and treat them with kinase inhibitors (or extracts from G1-phase cells) that induce chromosome de-compaction in vitro. Decondensed chromosomes will then be reconstituted with purified proteins that are implicated in establishing a specific interphase function. For example, using this new methodology we will ask how programs of gene expression re-established or initiated in interphase (by comparing transcription from the active and inactive X chromosomes) and how DNA is licenced for replication by the origin recognition complex (ORC), supplied with exogenous MCMs. We will employ advanced flow cytometry and microfluidic approaches to capture native mitotic chromosomes and then develop high-throughput assays to visualise and test chromosome structure. Collectively these studies will help to uncover the molecular mechanisms that underlie epigenetic inheritance as well as clarifying whether ‘modified’ mitotic chromosomes can used as a novel, experimentally tractable surrogate to model interphase.
References
1.Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction. Djeghloul D, Patel B, Kramer H, Dimond A, Whilding C, Brown K, Kohler AC, Feytout A, Veland N, Elliott J, Bharat TAM, Tarafder AK, Löwe J, Ng BL, Guo Y, Guy J, Huseyin MK, Klose RJ, Merkenschlager M, Fisher AG.Nat Commun. 2020 Aug 17;11(1):4118. doi: 10.1038/s41467-020-17823-z.PMID: 32807789
Djeghloul D, Dimond A, Cheriyamkunnel S, Kramer H, Patel B, Brown K, Montoya A, Whilding C, Wang YF, Futschik ME, Veland N, Montavon T, Jenuwein T, Merkenschlager M, Fisher AG.Nat Struct Mol Biol. 2023 Apr;30(4):489-501. doi: 10.1038/s41594-023-00943-7. Epub 2023 Mar 20.PMID: 36941433
3. Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.
Berger C, Premaraj N, Ravelli RBG, Knoops K, López-Iglesias C, Peters PJ. Nat Methods 20, 499–511 (2023). doi: 10.1038/s41592-023-01783-5. PMID: 36914814
Prof Dame Amanda Fisher | Biochemistry (ox.ac.uk)
For informal enquiries: amanda.fisher@bioc.ox.ac.uk; or lindsay.baker@bioch.ox.ac.uk
Project Code D9
Understanding and engineering microbial communities, including the human microbiome
Microbial communities contain many evolving and interacting species, which makes them difficult to understand and predict. The Foster lab combines molecular and cellular biology with ecological and evolutionary approaches to break down this complexity. Using both theory and experiment, we study how bacteria cooperate and compete in order to succeed in their communities. The lab also studies the ecological networks formed by interacting bacteria, with the goal of predicting and manipulating gut communities for better health. We have a range of projects on offer within the remit of the lab, which can be tailored to the student’s interests.
See our website for more details of our research: https://zoo-kfoster.zoo.ox.ac.uk/
Prof Kevin Foster | Biochemistry (ox.ac.uk)
For informal enquiries: Kevin.foster@biology.ox.ac.uk
Project Code D10
Available projects:
Molecular Characterisation of the Human Cold Shock Response: “Cooling the Cellular Clock”.
Despite the widespread use of controlled cooling in clinical and emergency settings, we know surprisingly little about the impact that cold temperatures have on human cells at the molecular level. We have discovered that cooling and subsequent rewarming triggers unexpected temperature specific responses in human cells, including changes to the chromosome architecture and the functioning of the circadian clock. The circadian clock is an important inbuilt time keeper that controls the alignment of cell physiology and organism behaviour with the earths day and night cycle. The DPhil project aims to unravel how cold induced structural changes, altered mRNA localisation and changes to mRNA metabolism, affect the workings and mechanics of the cellular clock.
Membrane less organelles and the spatial distribution of mRNAs in eukaryotic cells . Membrane less organelles (MLOs) are biological condensates of protein and RNA that are visible by microscopy as “droplet-like” structures in the nucleus and cytoplasm of all eukaryotic cells. Well-known eukaryotic MLOs include the nucleolus, stress granules and processing bodies. MLOs can form by assemblies of protein and RNA, creating distinct spherical liquid droplets. They play a key role in the subcellular organisation of the cellular space by creating distinct cellular compartments. Unlike membrane delimited organelles, MLOs can be dynamic and rapidly assemble or dissolve in response to specific cues. Whilst such RNA granules are common in cells, their function and how some mRNAs are selected whilst others are excluded, remains largely elusive. This project, using in vitro model condensates and in vivo approaches, aims to unravel the physical properties that determines the solvency of specific mRNAs for a particular condensate.
What do the projects offer?
The projects offer training in a wide range of state of the art methodologies including, super resolution microscopy, high through-put sequencing technologies, bioinformatics, in vitro mRNA production, tissue culture, cell biology and classic biochemistry and molecular biology techniques.
Recent key publication:
- Cold-induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm. Fischl H, McManus D, Oldenkamp R, Schermelleh L, Mellor J, Jagannath A, Furger A. EMBO J. 2020 Nov 16;39(22):e105604. doi: 10.15252/embj.2020105604
For informal enquiries: Andre.furger@bioch.ox.ac.uk
Associate Prof Andre Furger | Biochemistry (ox.ac.uk)
Project Code D11
Control of centrosome function during the cell cycle and beyond
Background:
Centrosomes are small cytoplasmic organelles with a wide variety of cellular roles; they nucleate microtubules, serve as mitotic spindle poles, template cilia formation, while also contributing to proteostasis, signalling, trafficking and organelle positioning. They are essential for proliferation of normal cells. Indeed, congenital mutations in centrosomal genes cause growth failure syndromes (1).
Centrosomes are membrane-free organelles that concentrate up to 200 different proteins into specific structures; each centrosome contains a pair of centrioles embedded in a pericentriolar matrix. These complex multiprotein assemblies undergo dynamic changes during the cell cycle (2, 3). For instance, a single new centriole is built on each old centriole during S phase, whereas the pericentriolar matrix, the main site of microtubule nucleation, undergoes expansion and remodelling before mitosis to boost microtubule production for spindle formation.
Phosphorylation-dependent regulation of centrosome components is likely to be vital for a range of centrosome functions, including microtubule nucleation. Although several kinases including Aurora-A, Nek2, Cdk1 and Polo-like kinases 1, 2 and 4, have been implicated in centrosome regulation, a comprehensive list of their targets in the organelle is still lacking. We are currently undertaking an unbiased proteomic survey to elucidate centrosomal targets for these kinases.
Aims:
The ultimate goal of this DPhil project will be to improve our understanding of how centrosome function (i.e. biogenesis, homeostasis and microtubule nucleation) is regulated during the cell cycle and in quiescence. First, we will functionally characterise newly identified phosphorylation sites in centrosomal proteins with an established role in these processes. By developing tools such as specific antibodies against phosphorylation sites along with cell lines where the sites are genetically edited, we will obtain mechanistic insight into how these post-translational modifications contribute to centrosome function. Second, we will ask if these phosphorylation sites are present across multiple cell types, and if so, whether their impact on centrosome function is the same. Results from our laboratory suggest that erythroid progenitors do not remodel their centrosomes during mitosis and therefore certain aspects of centrosome function are regulated in a cell type-specific manner (4). In these instances, post-translational modifications of centrosome components could play an even more important part. This hypothesis will be tested with the tools generated in the first aim, and proteomic survey of centrosomes from these unusual cell lineages will provide further insight. Finally, armed with knowledge from the first two aims, we will ask whether and how centrosomal microtubule nucleation capacity impacts on cellular processes such as trafficking, signalling and spindle formation.
Visit us on https://gergelylab.com
(1) Chavali, Putz and Gergely. 2014. The role of centrosomes in development and disease. Philos Trans R Soc Lond B Biol Sci.
(2) Tischer, Carden and Gergely. 2021. Accessorizing the centrosome: new insights into centriolar appendages and satellites. Current Opinion in Structural Biology.
(3) Vasquez-Limeta and Loncarek. 2021. Human centrosome organization and function in interphase and mitosis. Seminars in Cell & Developmental Biology.
(4) Tatrai and Gergely. 2022. Centrosome function is critical during terminal erythroid differentiation. The EMBO Journal.
For more information about the Gergely lab see: https://gergelylab.com
Dr Fanni Gergely | Biochemistry (ox.ac.uk)
For informal enquiries: fanni.gergely@bioch.ox.ac.uk
Project Code D12
Mechanisms of ubiquitin signalling in DNA repair
Research in the lab is focussed on understanding the mechanisms of ubiquitin signaling in DNA repair pathways, which are essential to maintain genome stability. The lab recently identified a novel unique deubiquitinase, called ZUP1, which dismantles a specific ubiquitin chains and has a putative role in DNA repair. However, there are still key gaps in our mechanistic understanding of ZUP1 that, as a lab, we aim to address using an integrated approach of biochemistry and cell biology. Promisingly, targeting DUBs with small molecule inhibitors is an emerging and attractive therapeutic avenue in cancer research, so this represents a cutting-edge area of biology with potential to benefit patients.
Through this DPhil project, you will build on our recent unpublished findings and further our mechanistic understanding of the cellular role of ZUP1 in DNA repair. We recently performed a genome-wide CRISPR-Cas9 screen to determine novel synthetic lethal interactions in cells with ZUP1 loss after DNA damage. We want to understand why these hits cause lethality in ZUP1 knockout cells and how we can leverage these findings to selectively kill cancer cells. To do this, we will use a range of advanced cell biology approaches (e.g. high-content single cell microscopy and individual replication fork analysis), CRISPR-Cas9 genome editing, DNA sequencing technologies, and ubiquitin proteomics tools available in the lab and wider Oxford environment. The results of this research will be essential to help us determine whether ZUP1 is a viable therapeutic target in cancer.
Dr Ian Gibbs-Seymour | Biochemistry (ox.ac.uk)
For informal enquiries from prospective students: Ian.gibbs-seymour@bioch.ox.ac.uk
Project Code D13
Structural studies of host-parasite interactions and structure-guided vaccine design.
Structural studies of host-parasite interactions and structure-guided vaccine design. Multiple possible projects.
For more information about the Higgins lab see: higginslab.web.ox.ac.uk
Prof Matt Higgins | Biochemistry (ox.ac.uk)
For informal enquiries: matthew.higgins@bioch.ox.ac.uk
Project Code D14
Tackling antimicrobial resistance using molecular dynamics and artificial intelligence
- It is very likely that the next pandemic will be due to pathogenic bacteria. While bacteria evolve at a rapid rate and are very quickly becoming resistant to currently available antibiotics, pharmaceutical companies are hesitant to invest in the development of novel antibiotics. Therefore, the burden of doing so, falls upon the non-profit sector, i.e. academics and charities. To this end we are using molecular dynamics simulations and artificial intelligence (AI) methods to understand the design principles required to develop novel, potent antimicrobial agents. We collaborate with experimental and computational groups within the UK and the USA including national laboratories in the USA.
- We are using modelling and simulations to design antimicrobial peptides which are then tested by collaborators, the results are fed into an AI model which in turn predicts a series of modified peptides which are then simulated. The key here is that the simulations provide mechanistic information. The student will be trained in state-of-the-art molecular modelling and simulation techniques and AI approaches and will have the opportunity to work in close collaboration with experimental colleagues.
To find out more about the Khalid lab see: https://khalidlab.web.ox.ac.uk/
Prof Syma Khalid | Biochemistry (ox.ac.uk)
For informal enquiries: syma.khalid@bioch.ox.ac.uk
Project Code D15
The energetics of protein import through the bacterial outer membrane
The asymmetric outer membrane of Gram-negative bacteria is not energised by an electrochemical gradient and no ATP exists in the periplasm. Consequently, essential energy-dependent processes at the outer membrane need to be driven by the proton motive force (PMF) across the inner membrane. The Ton and Tol systems of Gram-negative bacteria are able to convert the PMF into mechanical force at the outer membrane, which drive the import of nutrients and stabilises the outer membrane, respectively. These energy-transduction systems are important for bacterial survival especially during infection of hosts and so are viable targets for next-generation antibiotic design. However, we know very little about their molecular mechanisms of action. We recently developed a novel protein import assay that exploits the ability of bacteria to import very large molecules through the outer membrane by coupling to PMF-linked systems in the inner membrane. Moreover, we have developed this assay in a microfluidics/fluorescence microscopy format whereby import of single protein molecules attached to beads can be visualised in real time in living bacteria. This DPhil project will capitalise on these developments to explore the energetics of protein import across the bacterial outer membrane and determine how PMF-linked systems drive this import. The project would be particularly suitable for candidates with interests in molecular biophysics and microbiology. Some relevant recent outputs include: Mamou et al (2022) Nature 606, 953; Francis et al (2021) EMBO J., e108610; Gruszka et al (2020) Sci Adv. 6, eabc0330; White et al (2017) PNAS 114, 12051; Gruszka et al (2016) PNAS 113, 11841.
More information about the Kleanthous & Gruszka labs can be found at:
http://www.bioch.ox.ac.uk/research/kleanthous
https://kavlinano.ox.ac.uk/people/dominika-gruszka
Prof Colin Kleanthous | Biochemistry (ox.ac.uk)
For informal enquiries:
colin.kleanthous@bioch.ox.ac.uk
dominika.gruszka@physics.ox.ac.uk
Project Code D16
Discovering how epigenetics regulate gene expression in stem cells
Controlling how our genes are expressed is fundamental to cell function and development. While the molecular biology revolution of the mid-twentieth century defined the central dogma which states that DNA instructs the production of RNA and then protein, the mechanisms that unpin how the earliest steps of this cascade are controlled, namely how DNA is read and translated into the RNA remains very poorly understood. In eukaryotes, the ability to read DNA sequence is profoundly influenced by histones that package DNA into chromatin, and chromatin constitutes a central epigenetic regulator of RNA production and gene expression. However, our understanding of the mechanisms that enable epigenetic systems to control gene expression remain rudimentary at best. To address this fundamental problem, we use embryonic stems cells as a model to study how chromatin and epigenetic systems regulate gene expression to ensure stem cells remain pluripotent and can also support cellular differentiation and organismal development.
In the context of this overarching focus of the laboratory, a project is available to examine how proteins that function at CpG island elements (epigenetically defined elements associated with gene promoters in mammals) contribute to regulation of gene expression. The student will use and develop (via CRISPR-based genome engineering) embryonic stem cell lines containing degrons to rapidly deplete epigenetic regulators. They will then use genomic approaches coupled with next generation sequencing (ChIP-seq, RNA-seq, etc.) and/or live-cell imaging to determine how these systems affect chromatin modifications and gene expression. In particular, this will be applied to components of either the Polycomb repressive or Trithorax activator systems, which are paradigms of epigenetic gene regulation in mammals. As the project progresses there will also be an opportunity to dissect how these systems are used to regulate gene expression during cellular differentiation. The student will benefit from extensive support and training in the approaches necessary to tackle these fascinating problems, and will be at the forefront of uncovering how the epigenome shapes gene regulation and genome function.
(1) A CpG island-encoded mechanism protects genes from premature transcription termination. Hughes AL, Szczurek AT, Kelley JR, Lastuvkova A, Turberfield AH, Dimitrova E, Blackledge NP, Klose RJ. Nature Communications, 2023.
(2) Recycling of modified H2A-H2B provides short-term memory of chromatin states.
Flury V, Reverón-Gómez N, Alcaraz N, Stewart-Morgan KR, Wenger A, Klose RJ, Groth A. Cell, 2023.
(3) Distinct roles for CKM-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction. Dimitrova E, Feldmann A, van der Weide RH, Flach KD, Lastuvkova A, de Wit E, Klose RJ. Nature Structural and Molecular Biology, 2022.
(4) PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency. Dobrinić P, Szczurek AT, Klose RJ. Nature Structural and Molecular Biology, 2021.
(5) Live-cell single particle tracking of PRC1 reveals a highly dynamic system with low target site occupancy. Huseyin MK and Klose RJ. Nature Communications, 2021.
Prof Rob Klose | Biochemistry (ox.ac.uk)
For informal enquiries: rob.klose@bioch.ox.ac.uk
Project Code D17
Regulation of organelle homeostasis and dynamics by membrane contact sites
The eukaryotic cell harbour a set of membrane-bound organelle. Coordination of the activity within all of them is dependent on membrane contact sites. These contact sites serve as platform to exchange metabolites (such as lipids) and information between compartments. We study how membrane contact sites allow organelle biogenesis by providing the appropriate complement of lipid molecule for the build up of their membranes. We assess the effect of impairing these contact sites on organelle growth, functions and dynamics. To achieve these goals, we use protein structure prediction, high-end microscopy, functional genomics and mass-spectrometry-based lipidomics. Research projects are available in various directions. For instance, our recent discovery of a new structural interaction module between Miro, a key mitochondrial protein and a factor in Parkinson’s disease, and several interactors found often on different organelles paves the way to decipher the physiological role of this interaction, which will be tested by CRISPR-mediated genome editing. Another direction is about the effect of membrane lipid composition and membrane transporter activity. Membrane contact sites are sites of lipid transport and their dysfunction may cause aberrant membrane composition. Because membrane lipids are the solvent in which membrane transporter operates, it is expected that membrane composition should influence transporter activity. Indeed, through e genomics screen, we found indeed the first evidence that this could be the case. Interestingly, improper lipid transport between organelles led to decreased activity of plasma membrane transporters, which subsequently underwent endocytosis. A project is thus to study how membrane composition affects transporter activity, and what signal triggers their internalization. Several other projects are available and can be discussed with the candidates.
For more information about the Kornmann lab see: http://www.kornmann.group
Prof Benoit Kornmann | Biochemistry (ox.ac.uk)
For informal enquiries: benoit.kornmann@bioch.ox.ac.uk
Project Code D18
Maintenance of genome integrity through DNA repair
Preserving genome integrity through DNA repair is critical for human health and defects in these pathways leads to a variety of pathologies including neurodegeneration and cancer. Our research aims to understand the mechanistic basis of DNA repair, with specific reference to how a family of proteins called Poly(ADP-ribose) polymerases (PARPs) regulate these processes. Our long-term vision is to exploit this knowledge to treat a variety of diseases including neurodegeneration and cancer.
Research Projects
PARPs are a cornerstone of the DNA damage response that promote repair of breaks in the DNA helix by modifying proteins at the damage site with ADP-ribose - a process known as ADP-ribosylation. These pathways are critical to maintain genome integrity and are attractive clinical targets, with PARP inhibitors being used to treat breast and ovarian tumours. However, despite the success of PARP inhibitors in the clinic, the mechanistic basis of how PARPs regulate DNA repair is unclear, representing a fundamental barrier to refine and broaden the application of these agents in the clinic. The overall goal of our research is to address these fundamentally important questions by defining the mechanistic basis of how PARPs regulate a variety of DNA repair mechanisms. DPhil projects are available that integrate cutting edge genome engineering, proteomics and cell biology to address the following:
- How do PARPs become activated in response to DNA damage?
- How do these modifications regulate DNA repair?
- What regions of the genome are hotspots for PARP-dependent DNA repair.
Prof Nick Lakin | Biochemistry (ox.ac.uk)
For informal enquiries: Nicholas.lakin@bioch.ox.ac.uk
Project Code: D19
Cytoskeletal organisation of the malaria parasite
The malaria parasite, Plasmodium falciparum, infects various cell types within its insect and human hosts as part of a complex life cycle (1). In the human bloodstream, the parasite invades red blood cells to replicate inside, hidden away from the host immune system. To invade these cells, the parasite uses specialised invasive organelles, the rhoptries and micronemes (2).
The parasite cell needs to be precisely organised to successfully invade the host. The rhoptries and micronemes are held at the apical end of the cell, whereas its nucleus, mitochondria and other organelles are held toward the basal end (3). It is not known how the cell sets up and maintains this striking polarisation. One class of proteins proposed to contribute are the various cytoskeletal filaments that form fascinating structures throughout the parasite. This is exemplified by the microtubules, which form a beautiful array along the parasite membrane (4). However how these filaments link to other structures in the cell to carry out their function remains mysterious. Our lab is interested in how these filament systems position and shape organelles throughout the parasite cell. Our primary techniques are in vitro structural biology and biophysics, though we are expanding into in vivo techniques to examine these proteins within the malaria parasite itself.
The aim of this DPhil project is to further our understanding into how organelles are linked to the microtubule cytoskeleton in the malaria parasite. We will explore the following questions: We will identify and characterise novel microtubule-binding proteins to see how they link organelles to microtubules in vitro and in vivo. We will examine how they recruit organelles to the cytoskeleton using electron microscopy, then design assays to test their functional dynamics using biophysical techniques including TIRF microscopy. To dissect the protein function in cells we will use CRISPR/Cas9-based strategies to modify the protein inside the parasite. We will explore their localisation in cells and examine the consequences of knocking out the protein using light microscopy.
Please contact clinton.lau@bioch.ox.ac.uk,
For more information about the Lau lab see: https://laulab.web.ox.ac.uk/.
References
- Venugopal, K. et al. Nat. Rev. Microbiol. 2020 183 18, 177–189 (2020). https://doi.org/10.1038/s41579-019-0306-2
- Schrevel, J. et al. Parasitology 135, 1–12 (2008). https://doi.org/10.1017/S0031182007003629
- Fowler, R. E. et al. Parasitology 117, 425–433 (1998). https://doi.org/10.1017/S003118209800328X
- Ferreira, J. et al. bioRxiv (2022). https://doi.org/10.1101/2022.04.13.488170
Dr Clinton Lau | Biochemistry (ox.ac.uk)
For informal enquiries from prospective students: Clinton.lau@bioch.ox.ac.uk
Project Code D20
Toll-mediated regulation of intestinal stem cells
Toll-like receptors (TLRs) regulate the crosstalk between innate and adaptive immunity. They interact with evolutionary conserved pathogen-derived molecules but also with host ligands (damage/danger-associated molecules) that derive from injured tissues. This is probably why TLRs have been found to be expressed on several kinds of stem/progenitor cells including intestinal stem cells (ISCs). In some of these cells, the role of TLRs in the regulation of basal motility, proliferation, differentiation, self-renewal, and immunomodulation of SCs has been demonstrated.
However, the regulatory mechanisms by which TLRs may control ISC proliferation in normal conditions and in response to injury or infection are still unexplored. This is where the use of the vinegar fly Drosophila melanogaster can lead to faster progress. TLRs have been named after the identification of the TOLL gene in Drosophila more than 30 years ago, and the signalling pathway downstream of TOLL has been shown to be evolutionary conserved in innate immunity of flies, mice, and humans. Our published and unpublished work (the latter leading to the current proposal) has shown that the classical TOLL pathway is involved in the maintenance of gut bacteriome and in ISC renewal following infection. Our proposal is to identify the mechanisms by which TOLL is regulating ISCs and how downstream of TOLL, NF-κB and JNK orchestrate self-renewal and tissue regeneration.
Using the rich “toolbox” of Drosophila we will identify how inducing Toll activity in ISCs triggers cell division and what are the downstream targets of the Toll/NF-κB pathway. We know that one of these is JNK a signal that maintains “stemness” in the Drosophila gut. By conditional activation of the Toll ligand, Spaetzle (Spz), we will investigate whether it acts as a mitogen. Triggering Toll in ISCs activates Toll targets in other progenitor cell types as well as enterocytes and so either Spz or another cytokine-like molecule may act as a mitogen across the intestinal epithelium under stress or infection.
Prof Petros Ligoxygakis | Biochemistry
For informal enquiries: petros.ligoxygakis@bioch.ox.ac.uk
Project Code D21
Understanding the role of transporters & ion channels in health and disease
Transporters belonging to the Solute Carrier (SLC) family are integral membrane proteins that play essential roles in human physiology. They predominantly function to regulate the transport of small molecules into and out of cells and regulate the availability of amino acids, ions, sugars, lipids and vitamins within the body. SLC transporters also function within intracellular signalling networks, particularly with respect to amino acid and nutrient sensing. Recently my group discovered a new role of solute carriers in protein trafficking, revealing a hitherto unknown link between transport, cellular homeostasis and signalling in the cell (https://www.science.org/doi/10.1126/science.aaw2859 ).
My group has several projects suitable for DPhil students that focus on proteins linked to cancer drug discovery (see https://www.nature.com/articles/s41586-021-03579-z & https://www.nature.com/articles/s41467-021-27414-1 ), glycobiology & antifungal drug discovery (https://pubmed.ncbi.nlm.nih.gov/29143814/ ), lysosomal biology, metabolic and neurological diseases ( https://www.nature.com/articles/s41467-022-32589-2 ), antibiotic transport (https://www.science.org/doi/10.1126/sciadv.abh3355 ), antitubercular drug discovery (https://www.sciencedirect.com/science/article/pii/S0969212621002173 ) and trafficking within the cell.
DPhil students will be trained in single particle cryo-EM, fluorescence microscopy, antibody engineering, protein purification and the biochemical and biophysical characterisation of membrane proteins. Our research is moving into the fields of super resolution microscopy and in situ structural methods, including sub cellular fractionation, metabolomics and cryo-ET, in partnership with collaborators at the Oxford Parkinsons Disease Centre, NIH National Cancer Institute, MD Anderson Cancer Centre and Penn State University. Our goal is to understand and explain the role of solute carrier transporters in human health and disease.
Interested students are encouraged to reach out to either Prof. Newstead or Dr Parker for more information and discuss which project areas would match their research interests.
Prof Simon Newstead | Biochemistry (ox.ac.uk)
For informal enquiries: simon.newstead@bioch.ox.ac.uk or joanne.parker@bioch.ox.ac.uk
Project Code D22
Mechanisms of Wnt signal transduction across the plasma membrane.
The Wnt/b-catenin signalling pathway is essential in embryonic development and has important functions in tissue regeneration and overall maintenance of tissue homeostasis throughout the lifespan of multicellular organisms. The pathway is a highly orchestrated tug-of-war between two opposing multi-protein assemblies, the b-catenin destruction complex (DC) and the Wnt signalosome, that ultimately determines the fate of the transcriptional co-activator b-catenin. Furthermore, dysregulation of Wnt signalling is associated with many human diseases, most prominently cancer. Understanding how these pathways function at the molecular level is therefore of great importance.
Recent advances have significantly contributed to our understanding of the molecular mechanisms driving the destruction complex (Ranes et al 2021). However, much less is known about the mechanisms underlying the formation of the Wnt signalosome upon pathway activation.
In particular the mechanism(s) through which Wnt signals are transduced across the plasma membrane are still not well defined and intensely debated. The favoured models being: (I) Wnt acting as a molecular glue in the heterodimerisation of the Wnt-receptors FZDs and LRP5/6. (II) Wnt binding to the ectodomain of FZDs triggers allosteric conformational changes across FZD, independent of LRP5/6. However, we believe that these models are part of the same activation mechanism and that thus far these models have proven difficult to reconcile due to a lack of structural information of full-length FZDs and LRPs receptors complexes with and without Wnt.
The aim of the DPhil project is to gain structural insights into how the Wnt signal is transduced through the receptors into the cell. This will be addressed by single-particle cryo-Electron Microscopy (cryo-EM) reconstruction of in vitro reconstituted Wnt-receptor complexes of recombinant hFZD7, hLRP6 and hWnt3a proteins. The student will undertake different approaches to reconstitute Wnt-receptor (sub) complexes in either detergent-assisted or detergent-free environments and characterise these biophysically using SEC-MALS, differential scanning fluorometry, mass photometry and other techniques. The reconstituted subcomplexes will not only provide backup strategies but also different reagents to analyse by cryo-EM and structural proteomics methods such as crosslink mass spectrometry. With the reconstitution of FZD7-Wnt3a-LRP6 complex in a nanodisc lipid bilayer being the main structural target.
This project provides an excellent training opportunity in cutting-edge research and to contribute to advancing our understanding of a highly disease-relevant cell signalling process.
Dr Michael Ranes | Biochemistry (ox.ac.uk)
For informal enquiries: michael.ranes@bioch.ox.ac.uk
Project Code D23
What drives the evolution of epigenetic mechanisms?
Epigenetic regulation is fundamental to development as it establishes heritable changes in gene expression without affecting the underlying DNA sequence. Many epigenetic mechanisms exist, which specify gene expression states and have the potential to be transmitted through cell division. These epigenetic mechanisms are often widely conserved across eukaryotes. However, despite their ancient origin, many epigenetic mechanisms have been lost independently numerous times in different eukaryotic lineages. Why this happens is very puzzling. Recently we discovered a potential explanation for why one epigenetic pathway, cytosine DNA methylation (5-methyl-Cytosine), conserved from plants to humans, is frequently lost in different eukaryotes. We discovered that cytosine DNA methyltransferases can damage the DNA, introducing a form of alkylation damage, 3-methyl-cytosine, which is highly toxic (Rosic et al., Nature Genetics, 2018). This toxic effect results in the co-evolution of a DNA repair pathway, ALKB2, which is specialised to remove 3-methyl-cytosine from DNA. ALKB2 is present in species that have DNA methylation but absent from those that have lost the methylation machinery. Loss of ALKB2 may therefore predispose organisms to lose DNA methylation. This work acts as an example of how, more generally, epigenetic mechanisms might be lost due to negative consequences associated with them. In this PhD project we aim to identify other potential examples of costs associated with epigenetic mechanisms. To do this we will use co-evolution across species, using computational methods, to identify genes that are associated with particular epigenetic mechanisms across evolution. Importantly, we will then explore whether similar relationships are found in cancer, because epigenetic mechanisms are frequently lost or inactivated in cancer cells. The project will involve computational analyses of large datasets, in particular comparative genomics across eukaryotic species and gene expression analyses across multiple cancers, including single cell sequencing data analysis. The work will identify new explanations for why epigenetic pathways evolve, and link these to factors driving the evolution of cancer cells and the progression of the disease.
To find out more about the Sarkies lab see: https://psarkies.wixsite.com/epievo
Associate Prof Peter Sarkies | Biochemistry (ox.ac.uk)
For informal enquiries: Peter.sarkies@bioch.ox.ac.uk
Project Code D24
Interrogating nuclear structure-function relationships in mammalian cells by advanced super-resolution imaging
Three-dimensional (3D) chromatin organisation plays a crucial role in regulating mammalian genome functions such as RNA transcription, replication and DNA repair. Population-based sequencing approaches (e.g. Hi-C) have highlighted the compartmentalisation of chromatin into 0.5-1 MB sized topologically associating domains (TADs). However, many of the physical features at the single-cell level are still underexplored. Our primary research objective is to identify principles and underlying mechanisms of functional chromatin organisation in mammalian cells. Specifically, we aim to understand the interplay between biophysical forces, epigenetic memory, and cohesin complex activity to modulate cell-type-specific transcriptional programmes by directly visualising dynamic nuclear organisation and gene activity in living or 3D-preserved cells. To this end, we employ a combination of genetic editing with innovative in vivo/in situ fluorescence labelling and super-resolution imaging approaches. Our activities are closely linked to the Micron Oxford Advanced Bioimaging Unit and supported by our well-established ties to leading chromatin and epigenetic research groups within the Department and across Oxford.
For a MSc/PhD project, we seek (an) enthusiastic, proactive and adventurous student(s) eager to immerse in the latest imaging technologies to study topographical and biophysical aspects of gene regulation in an interdisciplinary environment. The topic of the project can be along the lines of either (1) studying single nucleosome dynamics within mesoscale chromatin domains using correlative single molecule tracking and super-resolution SIM imaging, (2) analysing loop-extruding and sister chromatid cohesive and loop-extruding cohesin complexes by super-resolution expansion microscopy (ExM) and/or super-resolution 3D correlative light and electron microscopy (CLEM), (3) studying the effect of directed phase separation on mesoscale domain organisation and transcriptional modulation, (4) examining mechanisms of gene reactivation during pathological de-differentiation processes (e.g. liver fibrosis, EMT), or (5) examining enhancer-promoter interactions e.g. in the alpha-globin locus, using multiplexed RNA-DNA-Immuno-FISH and correlative 3D super-resolution light end electron microscopy. The details of any project will be subject to personal preferences and be worked out closer to start date.
Main techniques: Mammalian tissue culture, molecular cloning, transfection, immunofluorescence labelling, fluorescence in situ hybridisation (DNA/RNA FISH), super-resolution structured illumination microscopy, single-molecule imaging, focussed ion beam scanning electron microscopy (FIB-SEM), computational image analysis.
Relevant papers:
Miron E, ..., Schermelleh L. 2020. Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin. Science Advances 6, eaba8811
Brown JM, … Schermelleh L, Buckle VJ. 2022. RASER-FISH, a non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protoc 17, 1306-1331.
Rodermund L,..., Schermelleh L#, Brockdorff N#. 2021. Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading. Science. 372: eabe7500
Ochs F, ..., Schermelleh L#, Lukas J#, Lukas C. 2019. Stabilization of chromatin topology safeguards genome integrity. Nature, 594: 571-574.
Schermelleh L et al. 2019. Super-resolution microscopy demystified. Nat Cell Biol 21: 72-84.
Associate Prof Lothar Schermelleh | Biochemistry (ox.ac.uk)
For informal enquiries: lothar.schermelleh@bioch.ox.ac.uk
Project Code D25
Protein structure and interactions in health and disease .
Our laboratory seeks to understand how protein functions arise from their molecular structure, conformational changes, and interactions, and how these processes are involved in human health and disease. Projects in at least three areas are possible: (1) We have a long-standing interest in the molecular mechanisms by which Influenza virus proteins function. To this end, we are studying the protein-protein and lipid interactions of ‘flu’ proteins to better understand their role in the virus life cycle and to identify potential therapeutic targets. (2) A collaborative project to combine experimental and computational tools to determine the timescale and energetics of conformational changes in proteins. (3) Investigations into the molecular mechanisms underlying chaperone protein activity, to explain how they interact with misfolded/unfolded clients.
A central technique of our laboratory is solution nuclear magnetic resonance (NMR) spectroscopy, which allows atomic-level studies of protein structures and their interactions. NMR can be uniquely informative in situations where the molecular conformations or interactions are dynamic or heterogeneous. However, we also use a wide variety of other biochemical and biophysical tools as needed for these investigations. We have collaborations with various research groups including virologists, cell biologists, and computational biologists.
Associate Prof Jason Schnell | Biochemistry (ox.ac.uk)
For informal enquiries: Jason.Schnell@bioch.ox.ac.uk
Project Code D26
Molecular mechanisms in brain development
Brain development relies on specialised cell surface proteins, the guidance receptors, which direct the migration of neuronal cells. These use context-dependent interactions with other proteins (ligands) to sense their environment. In this project, we focus on Unc5 guidance receptors and their FLRT ligands, which together with further binding partners, form combinatorial interactions that instruct the migration of neurons in early cortical development.
We will assemble a detailed spatiotemporal map of when/where different protein complexes form during radial migration, determine their atomic structures to obtain mechanistic insight, and design structure-based mutants to specifically target combinatorial ligand binding in cell biology and tissue models. The results will establish a molecular-level understanding of how radial migration is determined by these guidance receptors. This Wellcome-Trust funded project uses an integrated work package combining structural biology, protein engineering, proteomics, cell biology, advanced imaging and mouse technology. The project creates an outstanding interdisciplinary and international training environment for students to lead on a defined aspect of the project in the Seiradake lab, with the option to network and access training also through collaborating labs using different techniques. Project website: https://migrate.web.ox.ac.uk/
For more information about the Seiradake lab see: http://seiradake.web.ox.ac.uk
Prof Elena Seiradake | Biochemistry (ox.ac.uk)
For informal enquiries: elena.seiradake@bioch.ox.ac.uk
Project code D27
The molecular mechanism of establishment of sister chromatid cohesion at a single molecule resolution.
The error-free duplication and segregation of chromosomal DNAs is fundamental to cell proliferation. Sister DNAs generated during DNA replication must be held together from S phase until anaphase, when they finally disjoin to opposite poles of the cell. This process called sister chromatid cohesion (cohesion) ensures equal segregation of the genome. Cohesion results from entrapment of sister-DNAs within a highly conserved, ring-shaped protein complex called cohesin, with proteolytic cleavage of the cohesin rings being initiated prior to cell division to enable disjunction of sister chromatids. Cohesin is essential for orderly segregation of chromosomes during cell division, and when its function is perturbed, this can lead to human cancers and developmental disorders. However, despite the fundamental importance of cohesion for cell division, the mechanisms that control how cohesion is created remain enigmatic and constitute a major gap in our understanding of eukaryotic biology. Our research goal is to understand the molecular mechanism of sister chromatid cohesion. We have recently demonstrated that cohesin rings that trap unreplicated DNAs in G1 are ‘converted’ into cohesive molecules (trapping both sister DNAs) during replication (doi: 10.7554/eLife.56611). One can envisage two scenarios for how DNA associated cohesin rings become cohesive. 1. The replication machinery could simply pass through the large cohesin rings, resulting in entrapment of the replicated DNAs inside the rings. 2. Cohesin rings could be removed from ahead of the fork and deposited behind the fork (like the nucleosomes are). In order to test these two scenarios, we have recently set up a single molecule assay to track the fate of cohesin rings when they encounter the replication machinery on DNA (https://doi.org/10.1101/2022.09.15.508094) . This project will build upon this single molecule assay to test the requirement of the opening of cohesin rings 3 interfaces in order to establish cohesion. This will involve biochemical techniques like purification of human/Xenopus cohesin complex expressed in insect cells, fluorescent labelling of the purified complex and chemical crosslinking of any one or all 3 cohesin ring interfaces. This will be followed by single molecule imaging of replisome and cohesin. The in vitro single molecule assays will be complemented by in vivo analysis of single molecules of cohesin, we have recently begun single particle tracking of cohesin molecules inside living cells. The goal would be to observe cohesion establishment in vivo at a single molecule level. The in vivo work will involve CRISPR/Cas9 editing of human and DT40 cells to enable fluorescent labelling of cohesin complex and replisome components. The imaging will be done with a custom built TIR microscope as well as the ONI nano imager. We are a small, friendly, inclusive, supportive, and highly focussed group. As a Dphil/ MSc (res) student you will be very well looked after. Don’t hesitate to contact me if you need further information or a tour of the lab.
Dr Madhusudhan Srinivasan | Biochemistry (ox.ac.uk)
For informal enquiries from prospective students: madhusudhan.srinivasan@bioch.ox.ac.uk
Project Code D28
Understanding how bacteria adapt to stress, from single molecules to cell populations
Bacteria are extremely adaptable, which allows them to infect new hosts, evade immune defences, and survive antibiotic treatments. Under harmful conditions, bacterial cells induce protective stress responses and can acquire mutations that make them more stress resistant. Research in the Uphoff lab aims at understanding the interplay between phenotypic responses and genetic adaptation to stress. We have pioneered single-molecule and single-cell microscopy techniques that allow us to trace bacterial adaptation across enormous spatial and temporal scales, from cell populations down to individual molecular events. Equipped with these tools, our research addresses three main themes. Specific DPhil projects will be designed together with interested candidates.
Theme 1: Unravelling the unexpected complexity of bacterial stress responses
Studying bacterial stress responses at the level of single molecules and single cells has challenged the conventional wisdom of how these processes are regulated and what their functions are. We found that diverse types of stresses (e.g. reactive oxygen species, alkylating agents, antibiotics, etc), induce phenotypic heterogeneity in bacterial populations. We want to understand what causes this diversification of behaviour, and how it affects the adaptability of individual cells and the population.
Theme 2: Tracing the route from phenotypic tolerance to genetic resistance
Mutation is one of the most fundamental features of life, and we are acutely aware that human pathogens constantly mutate and evolve. Although DNA sequencing can identify adaptive mutations, much less is known about the molecular events that lead to such mutations appearing. We are developing experimental methods to trace adaptive mutations back to their molecular origins, which will help to predict and curb pathogen evolution. This is a great challenge because many processes in cells act in the creation or prevention of mutations, and various regulatory mechanisms exist that control them. Our lab innovated the use of microscopy to detect mutation events in real-time, which allows linking phenotypic and genetic changes in individual cells. Furthermore, using microfluidic growth devices, we can image thousands of individual bacteria simultaneously and monitor their phenotypes and fates under precisely controlled treatments over days. Mutations are often viewed as a “molecular clock” with a constant and uniform rate in all individuals. Instead, we found that mutation rates increase during stress and that intracellular noise causes cell-to-cell variation in mutation. We are now investigating if this means that evolutionary adaptation could be driven by subpopulations of cells with elevated mutation rates.
Theme 3: Understanding how bacteria adapt and survive within immune cells
Stress responses and tolerance mechanisms are particularly important for bacteria during infection. Phagocytes kill invading bacteria via a burst of reactive oxygen species that is thought to cause DNA damage. However, intracellular pathogens can withstand this damage and replicate within phagocytes. By adapting our single-molecule tracking approaches, we have succeeded in directly visualizing DNA repair functions in bacteria within live phagocytes. Using this approach, we can now investigate how intracellular bacteria survive the phagocyte immune defences.
Associate Prof Stephan Uphoff | Biochemistry (ox.ac.uk)
For informal enquiries: Stephan.uphoff@bioch.ox.ac.uk
Project Code D29
Evolution of chromatin across the tree of life
We are interested in how (and why!) chromatin evolved across the tree of life. What are the fundamental differences between chromatin in bacteria, archaea, and eukaryotes? Are there any? Why do eukaryotes only use histones as their principal chromatin protein? What’s so great about histones? Can we use proteins other than histones to build chromatin with similar properties? And can we imagine (and build!) a cell without chromatin whatsoever?
Our group combines computational (phylogenomics, structural modelling, functional genomics, machine learning) and experimental techniques (biochemistry, microbiology, genetics) to pursue these questions from multiple angles [1-4].
During this studentship, we want to tackle one of the following projects:
- Ultimate compaction. Histones are the principal building blocks of chromatin in eukaryotes but were generally thought to be absent from bacteria. We have recently discovered that this is not 100% true – there are some bacteria that encode and use histones to make chromatin [4]. One of these is the predatory bacterium Bdellovibrio bacteriovorus, which hunts and invades other bacteria. B. bacteriovorus is remarkable because their swimming “attack phase” cells are very small and somehow manage to condense an E. coli-size genome into a fraction of the volume [5]. We want to find out how they do this. Are histones involved? If not, what do the histones in these bacteria actually do?
- DNA glues. There are some proteins, like protamines in human sperm, that strongly compact DNA. They do so by virtue of being packed full of charged amino acids, notably arginine. Some bacteria also strongly compact their DNA (e.g. B. bacteriovorus, see above). Do they use similar proteins? For some species, like Chlamydia trachomatis, the answer appears to be yes [6]. For most others, we do not know. Do they encode their own unique toolkits? How do they manage the (often rapid) transition from a condensed to a decondensed state? This project will combine computational and high-throughput experimental approaches to characterize the repertoire and logic of these bacterial DNA glues.
What will you learn during your DPhil?
I am keen for students to master a broad range of tools, including both computational and experimental approaches. You can expect to learn how to culture and genetically manipulate a variety of microbes, to describe prokaryotic genome function using systems-level functional genomics approaches, and to analyze microbial evolution on a genome-wide scale.
- Rojec et al. Chromatinization of E. coli with archaeal histones. (2019) eLife 8:e49038
- Hocher et al. Growth temperature and chromatinization in archaea. (2022) Nature Microbiology 7:1932
- Stevens et al. Histone variants in archaea and the evolution of combinatorial chromatin complexity. (2020) PNAS 117:33384
- Hocher et al. Histone-organized chromatin in bacteria. (2023) bioRxiv https://doi.org/10.1101/2023.01.26.525422
- Sockett. Predatory lifestyle of Bdellovibrio bacteriovorus. (2009). Annu Rev Microbiol 63:523
- Barry et al. Nucleoid condensation in E. coli that express a chlamydial histone homolog. (1992) Science 256:377
My group will be joining the Department of Biochemistry in 2024. In the meantime, more information on the Warnecke lab can be found at: molsys.lms.mrc.ac.uk
For informal enquiries from prospective students: tobias.warnecke@bioch.ox.ac.uk
Project Code D30
Replication barriers and genome stability
A hallmark of ageing is the accumulation of genomic mutations and rearrangements through mistakes made during the normal processes of DNA replication, repair and chromosome segregation. It is thought that this gradual corruption of the genome results in gene regulatory changes, which cause cellular degeneration and functional decline that ultimately drives ageing and its associated diseases. Accordingly, the pace of genomic deterioration is likely to be a key determinant of healthy lifespan, which is strongly influenced by both environmental and genetic factors. Through a complete understanding of how mutations and genome rearrangements arise, as well as the factors that mitigate their occurrence, we will be better placed to develop new approaches to improve the healthy ageing of humankind.
Conflicts between replication forks and single-strand DNA breaks (SSBs) and protein-DNA complexes (PDCs) are a major threat to genome stability through their potential to cause fork collapse and failure of complete genome duplication. By exploiting state-of-the-art fission yeast genetics, advanced microscopy, protein biochemistry, advanced proteomics and genomic approaches, we aim to elucidate the different pathways that limit genome instability arising from replication fork- SSB/PDC conflicts, and how pathway choice is influenced by the nature and context of the SSB/PDC. This work will make a seminal contribution to our understanding of how genome deterioration, and consequent ageing and age-related disorders, is driven by problems that arise during S phase.
For more information about the Whitby lab see: https://whitbylab.com
Prof Matthew Whitby | Biochemistry (ox.ac.uk)
For informal enquiries: matthew.whitby@bioch.ox.ac.uk
