Supervisors and Projects

Project Code: M1

Resolving the rules of regulatory B cell (Breg) generation for therapeutic modulation in cancer and beyond

Immune system dysregulation underlies many diseases, leading to infectious disease susceptibility, cancer, immunodeficiencies, and autoimmune disorders. B cells, known for antibody production, also play crucial pro- and anti-inflammatory roles, with regulatory B cells (Bregs) shown to potently suppress immune responses. The importance of regulatory B cells (Bregs) has become clearly evident over the last decade. Bregs can suppress CD4 and CD8 T cell responses and are associated with poorer outcome across many cancer types(1). Conversely, Bregs are often dysfunctional/deficient in autoimmunity and checkpoint-inhibitor-associated toxicities(2). Functionally deficient B regulatory cells (Bregs) are linked to unfavourable clinical outcomes in autoimmune disorders such as Systemic Lupus Erythematosus, Rheumatoid Arthritis, and Multiple Sclerosis, and animal models support Breg therapy as an approach to treat these autoimmune disorders (3). The application of Breg cellular therapy to promote or suppress operational tolerance, depending on disease context, is an exciting prospect. Therapeutically targeting Bregs holds promise for restoring immunological health in autoimmune disorders and enhancing tumour recognition in cancers.

 However, little is known about human Bregs, including their generation, functional and molecular heterogeneity, and maintenance. The lack of phenotypic markers and the rarity of Bregs hinder research. Our work shows that IL10 and other Breg-associated genes are expressed at all B cell differentiation stages, varying by tissue type and disease, suggesting Bregs as a transient state rather than a specific lineage. This proposal leverages my preliminary work and integrates cutting-edge techniques in single-cell multi-omics (scMulti-omics, transcriptomics, proteomics, B cell receptor) and cell culture to investigate human Breg biology. This promises to significantly advance Breg understanding and identify Breg-specific drug targets for cancer and autoimmune diseases.

In aim1, you will characterise the diversity, function and co-dependences of naturally occurring in vivo and ex vivo generated human Bregs from existing scMulti-omics datasets, and predict key signals (transcription factors (TFs) and pathways) associated with Breg generation.

Aim 2 will resolve factors influencing the dynamics of ex vivo Breg generation, plasticity, function and stimulation via cell culture, transcriptomics and temporal modelling. You will then develop a novel experimentally-informed artificial intelligence (AI) model to decipher the complex relationships between factors, making this problem conceptually tractable. This open-source model will become a platform for the community to contribute to, including the effect of different experimental conditions and drugs.

Finally, in aim 3 you will confirm the signals required for Breg generation which will be used to predict drugs that impact generation of Bregs in vitro. This will be achieved through gene knock-down and over-expression experiments of predicted TF and key receptors derived from scMulti-omics data analyses in aim 1 to finally confirm drug targets that influence dynamics and/or stability of Breg generation in vivo.

This project couples the standardisation of experimental protocols and AI approaches to open up the field of Breg biology and will make experimentation interpretable for drug development prior to clinical translation.

Associate Prof Rachael Bashford-Rogers | Biochemistry (ox.ac.uk)

For informal enquiries: rachael.bashford-rogers@bioch.ox.ac.uk

Project Code M2

Role of transposable elements in development to regulate gene expression

Transposable elements are mobile genetic elements that make up 50% of the mammalian genome. Transposable element mobility is dependent on their ability to produce a full-length transcript. This means, TE expression is not only dangerous due to transposition events, but also due to activation of regulatory sequences with the potential to influence gene expression.

Because of the thread to genome integrity, cells have evolved a range of repressive mechanisms to prevent their expression. Epigenetic modifications such as DNA methylation and histone 3 lysine 9 trimethylation (H3K9me3) are particularly important. While older elements – more sequence diverse elements - have been linked to sequence-specific repression by Krüppel associated box (KRAB) zinc-finger proteins younger elements – with high sequence identify - tend to be silenced by the HUSH complex via H3K9me3. As a result, most TEs are transcriptionally silenced and not expressed in somatic cells. Interestingly, TE expression is essential for embryonic development. In mice, knock-down of specific TE families results in failure of embryos to progress past the 2-cell stage. Only 1-2% of TEs are young, full length, and still capable of transposition. However, evolutionarily young elements have highly similar sequences compared to older, more divergent TEs. Due to this, transcripts from young TEs cannot be mapped to their locus of origin with confidence using conventional short-read sequencing. Hence, detected expression could either be from a small proportion of young TE loci, or from all elements in a subfamily. We developed a long read RNA sequencing techniques called CELLO-seq to overcome these limitations. CELLO-seq enables us to map TE transcripts to their original genomic locus. As CELLO-seq also provides single-cell resolution, it allows us to determine TE expression heterogeneity between single cells in the embryo. Our work makes use of embryonic stem (ES) cell-based models to study molecular mechanisms by which Transposable elements influence gene expression. We use CRISPR/Cas9 based genome engineering to establish defined genetic backgrounds to test specific questions. We use next generation sequencing (NGS) and third generation sequencing (TGS) long read sequencing-based methods extensively, for example to map specific histone/DNA modifications and other chromatin features and to establish locus-specific transposable element expression. We make use of retrotransposition assays and flow cytometry based approaches to assess the role of transposon mobility in cells. We also use single molecule tracking approaches to investigate the dynamics of Transposable element RNA and its interaction with chromatin in early development. 

Dr Rebecca Berrens | Biochemistry (ox.ac.uk)

For informal enquiries: rebecca.berrens@bioch.ox.ac.uk

Project Code M19

Structural biology of DNA topology remodelling machines

Genetic information is stored in spectacularly large and thread-like molecules, the chromosomal DNA. This poses the fundamental “spaghetti problem”: DNA entanglements must be prevented by creating structured entities called nucleoids or chromosomes. Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases solve this issue in most if not all organisms.

We are interested in:

1. How ring-like SMC complexes use the power of ATP hydrolysis to extrude large DNA loops
2. How SMC complexes cooperate with DNA topoisomerases to prevent DNA entanglements
3. How pathogens such as bacteriophages and infective plasmids manipulate host chromosomes, and interact with host defences that use the mechanisms above

We address these questions using biochemical reconstitution, cryo-EM, and advanced bacterial genetics. We capture bacterial and viral genome remodelling machines in action, aiming to directly visualize how they work. Our long-term goal is a full mechanistic understanding of the processes that control the three-dimensional structure of chromosomes across the tree of life.

Dr Frank Bürmann | Biochemistry (ox.ac.uk)

For informal enquiries: frank.burmann@bioch.ox.ac.uk

Project Code M3

Single-molecule studies of chromatin replication

The copying of DNA, or DNA replication, is essential to cellular function. In eukaryotes it is carried out with high fidelity by a multi-protein complex called the replisome. Failure to do so can lead to defects in the copied genome, slow down or stall the replisome, and result in genomic instability or cell death. Mechanistic insight into normal replisome functioning is a prerequisite for understanding such failure and its downstream consequences.

This is an exciting time in eukaryotic DNA replication, as advances in genetics, biochemistry, and structural biology have identified the essential replisome components together with their stable arrangements. However, in operation the replisome is an active molecular machine with transient parts. Thus, a full mechanistic insight into this molecular machine requires a description of its dynamics. Because the replisome is active on DNA that is compacted into chromatin, this description must include the duplication and reassembly of all DNA-associated proteins. Understanding the coupling between these processes has fundamental implications for epigenetic inheritance and cancer.

Here, using an integrated approach at the interface of single-molecule biophysics and biochemistry, you will help to gain biophysical understanding of the operation of an individual eukaryotic replisome by examining its functioning on DNA and/or chromatin. In doing so, you will acquire a broad set of skills at the interface of single-molecule biophysics and biochemistry. You will start by first become familiar with the underlying molecular biology and biochemistry and learn to perform high-quality experiments in this context. You will then be introduced to state-of-the-art single-molecule techniques such as integrated force-fluorescence microscopy and TIRF microscopy. It is expected that using the combination of these biochemical and biophysical approaches you will gather data to examine several aspects of replisome functioning at the ensemble and single-molecule levels. To quantify the data that you will acquire, you will be introduced to and asked to make use of several forms of data analysis. The outcome of the project will contribute to new models of eukaryotic DNA replication that include a focus on its dynamic aspects.

Prof Nynke Dekker | Biochemistry (ox.ac.uk)

For informal enquiries: nynke.dekker@physics.ox.ac.uk

Project Code M4

Chromosome associated mitotic inheritance and DNA methylation

Understanding how DNA methylation impacts chromosome structure and regulates epigenetic inheritance is important for understanding tissue specific gene regulation, genomic imprinting and X-inactivation. We will take a three-pronged approach to defining how 5-methylcytosine (5mC) and the factors that bind to this modification, influence cellular memory. Firstly, we will examine the physical and structural properties of metaphase chromosomes isolated from wildtype or DNA methylation-depleted (or degron-targeted) equivalents, using CryoET and optical tweezer approaches. This work will done in collaboration with Dr Lindsay Baker (Oxford Biochemistry) and Professor David Rueda (Imperial College London). We will quantitatively assess chromosome-associated proteins and RNAs that are enriched in each of these samples to identify candidate 5mC-dependent mitotic bookmarking factors. Finally, we will use this information to determine whether genomic imprinting, X-chromosome silencing or lineage-restricted gene expression, is perturbed by manipulating 5mC-dependent factors (either alone, or with other chromatin modifiers) during mitosis. This study will inform cell reprogramming strategies and may help in understanding how age-related changes in DNA methylation and histone H3K9me3 erode cellular memory.

References 

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

Djeghloul, D., Dimond, A., Cheriyamkunnel, S. et al. Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis. Nat Struct Mol Biol 30, 489–501 (2023). https://doi.org/10.1038/s41594-023-00943-7

Dimond, A., Gim, D.H., Ing-Simmons, E. et al. PBK/TOPK mediates Ikaros, Aiolos and CTCF displacement from mitotic chromosomes and alters chromatin accessibility at selected C2H2-zinc finger protein binding sites. (2024). https://doi.org/10.1101/2024.04.23.590758

Berger C, Premaraj N, Ravelli RBG, Knoops K, López-Iglesias C, Peters PJ.

Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.

Nat Methods 20, 499–511 (2023). doi: 10.1038/s41592-023-01783-5. PMID: 36914814

Meijering AEC, Sarlós K, Nielsen CF, Witt H, Harju J, Kerklingh E, Haasnoot GH, Bizard AH, Heller I, Broedersz CP, Liu Y, Peterman EJG, Hickson ID, Wuite GJL.

Nonlinear mechanics of human mitotic chromosomes.

Nature. 2022 May;605(7910):545-550. doi: 10.1038/s41586-022-04666-5. Epub 2022 May 4.PMID: 3550865

Prof Dame Amanda Fisher | Biochemistry (ox.ac.uk)

For informal enquiries: amanda.fisher@bioch.ox.ac.uk

Project Code M5

The role of the centrosome in the spatiotemporal control of mitosis

General Background

In our laboratory we are fascinated by the molecular mechanisms that enable and control cell division. In particular, we want to understand how cells minimise chromosome mis-segregation events during mitosis to maintain their genome integrity. Proper chromosome segregation requires the formation of a functional spindle, composed of microtubules and associated proteins. Microtubules are focused in two spindle poles, organized by the two centrosomes.

Centrosomes are small cytoplasmic organelles that not only serve as mitotic spindle poles but also template cilia formation and contribute to signaling, trafficking, and organelle positioning. They are essential for normal cell proliferation; congenital mutations in centrosomal genes lead to various growth failure syndromes. Like DNA, centrosomes undergo semi-conservative duplication in S-phase, a process that ensures the presence of two functional centrosomes in cells when they enter mitosis.

Project Background

Aurora-A is a crucial protein kinase involved in centrosome maturation, mitotic entry, and mitotic spindle formation. Each role requires a specific binding partner, with interactions mediated by intrinsically disordered regions (IDRs)—protein domains that lack a stable three-dimensional structure. Despite their lack of defined structure, IDRs play important roles in various biological processes, including signaling pathways and transcriptional regulation, and are often mutated in diseases. Aurora-A has long been recognized as an oncogene, and its inhibitors are being explored for cancer treatments.

Recent results from our group suggest that centrosomes play a crucial signaling role by concentrating and activating Aurora-A kinase. In fact, the centrosome appears to be the only site in mitotic cells that can generate large amounts of T loop-phosphorylated (active) Aurora-A. The active kinase then forms a complex with TPX2 on the mitotic spindle and phosphorylates several important substrates. If Aurora-A skips centrosomal recruitment, it can still bind TPX2, albeit much less efficiently. Furthermore, the TPX2:Aurora-A complex, which uses non-phosphorylated Aurora-A, can still target some known substrates for phosphorylation but not all. This drop in kinase efficacy leads to spindle abnormalities and chromosome segregation errors.

Project Details

The primary aim of this project is to understand the rules governing Aurora-A's interactions with its substrates in cells.

Specifically, we aim to answer the following questions:

• Why do certain substrates, like the spindle pole protein NuMA, require Aurora-A activated at the centrosome, while others do not?
• When does centrosomal Aurora-A signal generation begin and end in cells, and can we modulate Aurora-A activity by altering this signalling window?
• How does Aurora-A (or the Aurora-A:TPX2 complex) differentiate between these substrates?
• Does the subcellular localization of the kinase and/or the substrates affect phosphorylation efficiency?

To address these questions, we will use various cell and molecular biology tools, along with a chemical biology toolkit developed through a multidisciplinary collaboration. The latter will allow us to target protein-protein interactions with advanced peptide-based inhibitors. Results may necessitate subsequent identification of new Aurora-A-binding partners and substrates using proteomic approaches.

The project will provide experience in a wide range of techniques, including:

• Gene editing
• Molecular and cell biology
• Biochemistry
• High-resolution microscopy
• Proteomics

This project will help us understand the mechanisms controlling cell division and explore the potential therapeutic applications of targeting Aurora-A interactions in cancer treatment.

References:

• Holder, J*, Miles J* et al.,Bayliss, R⁺ and Gergely, F⁺ (2024) CEP192 drives spatio-temporal control of mitotic Aurora-A.
• Carden, S*, Vitiello, E*, Rosa e Silva, I, Holder, J, Quarantotti, V, Kishore, K, Roamio Franklin, VN, D'Santos, C, Ochi, T⁺, van Breugel, M⁺ and Gergely, F⁺ (2023). Proteomic profiling of centrosomes across multiple cell and tissue types by a new affinity capture method. Dev. Cell .
• Burgess, S.G., Mukherjee, M., Sabir, S., Joseph, N., Gutiérrez-Caballero, C., Richards, M.W., Huguenin-Dezot, N., Chin, J.W., Kennedy, E.J., Pfuhl, M., Royale, S.J., Gergely, F. & Bayliss, R. (2018) Mitotic spindle association of TACC3 requires Aurora-A-dependent stabilization of a cryptic α-helix. EMBO J. 37 (8), doi: 10.15252/embj.201797902
• Richards, M.W., Burgess, S.G., Poon, E., Carstensen, A., Eilers, M., Chesler, L. & Bayliss, R. (2016) Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors. Proc. Natl. Acad. Sci. USA.113, 13726-13731.
• Review article: Ali A and Stukenberg TP (2023) Aurora kinases: Generators of spatial control during mitosis. Front Cell Dev Biol Mar 13:11:1139367.

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 M14

From bacteriocins to bacteriophages: structural biology of bacterial molecular machines

Bacteria thrive in different environments, adapting to the competition, predation, and fluctuating nutrients availability. Their diversity produces fascinating examples of protein machines with unique enzymatic activities.

Research in Ghilarov lab aims to reveal fundamental principles of how bacterial molecular machines use energy to manipulate three-dimensional structure of peptides and nucleic acids. Understanding these principles allows to control the activities of molecular machines, and ultimately to design artificial nanoscale devices. We are using a combination of cryoEM, X-ray crystallography, biochemistry, genetics and chemical biology approaches and collaborate with experts in modelling, protein design and single-molecule methods. Specific DPhil & MSc projects will be tuned to candidates’ interests, but must be related to one of the three lab themes:

Theme I: Ribosomally synthesized post-translationally modified peptides (RiPPs)

RiPPs enable bacterial competition, virulence, and defense against bacteriophages. RiPPs are genetically encoded, and converted into bioactive molecules by multienzyme complexes. Harnessing these complexes’ ability to modify custom peptides will allow synthesis of engineered molecules with programmed sequence. We are working on  different RiPP systems, including LAPs, lasso-peptides and thiopeptides, to get insights about RiPP biosynthesis, their ecological role, and to engineer them to create new-to-nature natural products[1,2].

Theme II: Bacterial DNA topoisomerases

Bacterial type II topoisomerases gyrase and topoisomerase IV are essential for removing positively supercoiled DNA in front of the progressing polymerases, introducing negative supercoiling required for chromosomal homeostasis, and segregating daughter chromosomes after replication. They work in close connection with the replisome and SMC proteins, directly and indirectly affecting all genomic transactions in the cell. The research in the lab aims to address a fundamental question of how topoisomerases use energy of ATP to introduce defined topology in DNA. We have developed cryoEM approaches to study gyrase and topoisomerase IV, resulting in a determination of the first gyrase structure in complex with chirally wrapped DNA[3]. We are also interested in protein and small molecule inhibitors of topoisomerases as inspiration for a new generation of antibacterial drugs[4,5], and in mechanisms of resistance to antibiotics related to topoisomerases.

Theme III: Bacterial antiviral defense systems

The field of bacterial phage immunity is rapidly expanding and is one of the most exciting topics of contemporary microbiology. We are studying a complex defense system called BREX (bacteriophage exclusion system). BREX components are dissimilar to any other proteins, and their functions cannot be easily predicted or deduced. Many of them bear similarity to eukaryotic proteins such as ORC/Cdc6. BREX complex assemble on DNA in a megadalton-size multiprotein complex, preventing phage replication by an unknown mechanism. We have recently determined structures of methytransferase BrxX[6] responsible for the self/non-self discrimination within the complex, and are actively investigating other components and homologous systems.

[1] Ghilarov et al. Mol Cell (2019) https://doi.org/10.1016/j.molcel.2018.11.032

[2] Travin et al. JACS (2018) https://doi.org/10.1021/jacs.8b02277

[3] Michalczyk et al. bioRxiv (2024) https://doi.org/10.1101/2024.04.12.589215

[4] Bakker et al. Nat Chem (2024) https://doi.org/10.1038/s41557-024-01516-x

[5] Michalczyk et al. Nat Catal (2023) https://doi.org/10.1038/s41929-022-00904-1

[6] Drobiazko et al. bioRxiv (2024) https://doi.org/10.1101/2024.04.12.589305

Ghilarov lab website: ghilarovlab.com

Contact email address for informal enquiries from prospective students: Dmitry.ghilarov@jic.ac.uk

Project Code M6

Targeting DNA repair mechanisms in precision cancer therapies

Cancers often have defects in processes that repair damage to their genetic material and tumours survive by becoming ‘addicted’ to processes that compensate for loss of these mechanisms. This is an Achilles heel of the tumour and using drugs to interfere with these pathways kills cancer cells. Principal in this strategy is using small molecule inhibitors of PARPs, a family of enzymes that modify proteins with ADP-ribose to promote DNA repair. PARPs are critical for the survival of cells with defects in the BRCA1 and BRCA2 breast and ovarian cancer genes and PARP inhibitors are used to kill these tumours.

However, despite the success of PARP inhibitors in the clinic, the effectiveness of PARP inhibitors differs between patients and tumours develop resistance to this treatment. The mechanistic basis of how PARPs regulate DNA repair is unclear, representing a fundamental barrier to addressing these challenges. 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 and MSc projects are available that integrate cutting edge genome engineering, proteomics and cell biology to address the following:

a) How do PARPs become activated in response to DNA damage?

b) What proteins do they modify at sites of DNA damage?

c) How do these modifications regulate DNA repair?

These multidisciplinary hypothesis-driven research projects will increase our understanding of how PARPs regulate DNA repair and provide critical information to develop novel strategies that target PARPs to treat a variety of pathologies

Prof Nick Lakin | Biochemistry (ox.ac.uk)

For informal enquiries: nicholas.lakin@bioch.ox.ac.uk

Project Code M7

Toll signalling in Drosophila intestinal regeneration

Brief description of project(s) or research theme (no more than 500 words): The gut is an interphase where epithelial renewal and microbiota maintenance are balanced with immunological regulation. How these functions integrate with cellular signalling is unclear. In the Drosophila gut, asymmetric mitosis of Intestinal Stem Cells (ISCs) produces Enteroblasts (EBs) that differentiate to Enterocytes (ECs). By using cell-specific RNAi we found that the immune receptor Toll, is needed for ISC mitosis in homeostasis and infection. Conversely, Toll gain of function in ISCs and EBs pushed ISCs into mitosis to produce EBs but blocked EB to EC differentiation, resulting in intestinal dysplasia. Toll activity was mediated by the classical Toll pathway components, JNK and Akt/mTOR signalling. These phenotypes were independent of the microbiota as seen in germ free flies. However, Toll activation resulted in increased density of commensal gut bacteria by transcriptional suppression of host lysozymes and amidases. These results indicate Toll as a major evolutionary conserved regulator of the intestinal landscape. The project will extend on these results and delve deeper on the metabolic requirement of ISC mitosis as well as the role of microbiota as a means to prevent the establishment of an enteric pathogen.

Prof Petros Ligoxygakis | Biochemistry

For informal enquiries: petros.ligoxygakis@bioch.ox.ac.uk

Project Code M8

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 M9

Protein structure and interactions in health and disease.

Our laboratory seeks to understand how protein functions arise from their molecular structure and interactions, and how these processes are involved in human health and disease. Projects in at least two areas are possible. (1) We have a long-standing interest in the molecular mechanisms by which Influenza virus proteins function. 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) More recently, we have begun to study the structure and function of chaperone proteins, especially J-domain proteins, to understand how they interact with misfolded/unfolded clients and with the HSP70 machinery.

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 M10

Structural biology of brain development

How does a single cell give rise to the amazingly complex human brain?

Brain development hinges on the proliferation and self-assembly of cells to form highly organized brain tissues and circuits. This intricate process relies on specialized receptor proteins that orchestrate the movements of brain cells during development. These receptors, situated on the cell surface, function like a cellular navigation system: they detect various cues (ligands) in the cellular environment and initiate signaling cascades that guide cells in the right direction. Collectively, these receptor-ligand interactions ensure the correct positioning of cells and their axons/dendrites within brain tissue, thereby coordinating the formation of brain circuits.

Approach

Our lab is part of a network that adopts an interdisciplinary approach to understand molecular functions within complex tissues. We specialize in structural biology (especially cryo-electron microscopy and X-ray crystallography) and advanced cell biology techniques. We integrate our findings with cryo-electron tomography, in vivo models, and super-resolution fluorescence microscopy through close collaboration with world-leading experts in Oxford (Baker), Barcelona (del Toro), and Bordeaux (Naegerl).

Outcome

By leveraging our established experimental pipeline, you will lead an interdisciplinary project aimed at unraveling how specific receptor-ligand interactions direct brain development. This project will yield groundbreaking discoveries about the molecular mechanisms underlying brain formation and function, much of which remains unknown. The findings will be directly relevant to brain disorders associated with these receptors. The project offers training in multiple techniques, particularly in structural biology and cell biology. Additionally, you will benefit from regular meetings with collaborators, providing further training and networking opportunities. Our team frequently engages in hands-on collaborative work in other labs within MIGRATE: https://migrate.web.ox.ac.uk/.

Prof Elena Seiradake | Biochemistry (ox.ac.uk)

For informal enquiries: elena.seiradake@bioch.ox.ac.uk

Project Code M11

Understanding molecular mechanism that transcription of the non-coding genome  

Recent technological advances have revealed a plethora of diverse long non-coding (nc) RNA molecules produced from eukaryotic genomes. Mutations in non-coding regions of the genome and altered expression of ncRNAs underpins a number of pathologies including cancer. Yet, very little is known about mechanisms involved in production of ncRNAs preventing us from understanding their role in health and disease. Our previous work lead to discovery that in contrast to mRNAs, nc transcripts rely on distinct and poorly understood mechanisms that control their RNA polymerase II (Pol II) transcription. As a result, ncRNAs are non-polyadenylated and targeted by the cellular RNA degradation machinery, RNA exosome.

The PhD project aims to fill the key gaps in our understanding of the transcriptional mechanisms involved in regulation of ncRNA. This will be achieved through the Aims 1-3. A PhD student will identify and characterise transcription complexes linked to production of ncRNA biochemically (Aim 1) and investigate how these complexes are recruited to Pol II during transcription and how they control biogenesis of ncRNA in human cells using state-of-the-art genomic approaches (Aim 2 and 3).

Associate Prof Lidia Vasilieva | Biochemistry (ox.ac.uk)

For informal enquiries: Lidia.vasilieva@bioch.ox.ac.uk

Project Code M12

DNA 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

Project Code M13

Molecular mechanisms underlying viral evolution and host changes

We seek to understand how enveloped viruses evolve and use this knowledge to come up with ways to deal with future pandemics.

Many deadly human pathogens, such as influenza and SARS viruses, are made up of just a few components but can infect a number of different hosts. How is it possible that these components suffice to fulfil all the functions necessary for a virus to infect the cell and then to assemble into a new viral particle? How do viral proteins perform multiple functions and how does the virus manage to retain all these functions as it evolves? How does a virus infect different hosts using the same set of its own proteins to engage a range of machineries of different hosts? And, finally, how does a virus evolve and ‘learn’ to optimise its interactions with a new host?

The recent COVID-19 pandemic has demonstrated that these questions are key to understand where new viruses come from and how they evolve upon transmitting to a new host. We have used biochemical, biophysical, and structural methodologies, mainly cryoEM, to reveal the mechanisms by which SARS-CoV-2 became able to infect humans and then further evolved to optimise viral infectivity in the variants of concern. [1-7]

The lab continues to work on coronaviruses and studies influenza viruses to understand how their proteins achieve the versatility needed to infect diverse hosts and fulfil multiple functions during infection. In particular, we want to explain how related viral strains use similar glycoproteins to engage receptors as different as glycans and proteins. We are also interested in understanding how viral proteins come together during virus assembly: how do they find other viral components, how do they ‘coerce’ the host to transport them, and how do they exclude hosts proteins from growing viral particles.

Tackling these questions can directly impact public health. The more we understand the rules governing the evolution of viral proteins, the better we can predict the impact of emerging viruses and thus increase our pandemic preparedness. Our long-term aim is to use structural and mechanistic insights to guide design of much-needed new antivirals and vaccines against zoonotic viruses.

Several projects encompassing areas above are available in the lab. If you are interested, please apply and do not hesitate to get in touch earlier to discuss more details. I am committed to working with you to scope projects that combine your interests in specific research questions as well as methodologies, your long-term career aspirations, and available expertise and pilot data in the lab. In this way we will devise the MSc or DPhil project that bests suit you.

REFERENCES (#co-first, •corresponding):

1. Antoni G. Wrobel# (2023) “Mechanism and evolution of human ACE2 binding by SARS-CoV-2 spike” Current Opinions in Structural Biology 102619.

2. Valeria Calvaresi#, Antoni G. Wrobel#, Joanna Toporowska, Dietmar Hammerschmid, Katie J. Doores, Richard T. Bradshaw, Ricardo B. Parsons, Donald J. Benton, Chloë Roustan, Eamonn Reading, Michael H. Malim, Steven J. Gamblin, Argyris Politis#. (2023) “Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein” Nature Communications, 14 (1), 427

3. Antoni G. Wrobel*#, Donald J. Benton*#, Chloë Roustan, Annabel Borg, Saira Hussain, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin# (2022). “Evolution of the SARS-CoV-2 spike in the human host” Nature Communications 13, 1178.

4. Antoni G. Wrobel*#, Donald J. Benton*#, Pengqi Xu, Annabel Borg, Chloë Roustan, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin# (2021). “Structure and binding properties of Pangolin-CoV Spike glycoprotein inform the evolution of SARS-CoV-2.” Nature Communications, 12 (1), 837.

5. Donald J. Benton*#, Antoni G. Wrobel*#, Chloë Roustan, Annabel Borg, Pengqi Xu, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin#. (2021) “The effect of the D614G substitution on the structure of the spike glycoprotein of SARS-CoV-2” Proceedings of the National Academy of Sciences, 118(9), e2022586118.

6. Donald J. Benton*#, Antoni G. Wrobel*#, Pengqi Xu, Chloë Roustan, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin# (2020) “Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion” Nature 588(7837), 327–330.

7. Antoni G. Wrobel*#, Donald J. Benton*#, Pengqi Xu, Chloë Roustan, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin#  (2020) “SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects.” Nature Structural and Molecular Biology 27, 763–767 (2020).

For informal enquiries: 

Current: antoni.wrobel@crick.ac.uk

From 01/09/24: antoni.wrobel@bioch.ox.ac.uk

Project code D1

Building models of infection and commensalism for electron cryotomography

This research aims to understand how bacterial responses to other cells and small molecules such as antibiotics depend on their environment.  Due to their size, bacteria can be challenging to study by light microscopy.  However, electron cryotomography (cryoET), which uses frozen hydrated samples to capture native structural details about cells, viruses and organelles at molecular resolution, is perfectly suited for imaging at this scale.   In this project, we will adapt co-culture protocols for bacteria and mammalian cells to be suitable for cryoET.  We will use a combination of mammalian growth factors and coatings with a solid medium approach for bacteria growth developed in the lab to produce spatially stabilised co-cultures suitable for cryoET and fluorescence microscopy.  Initially, we will focus on models of bacterial infection and commensalism in the gut including immune and epithelial cells, but there is potential to be comparative with other microbial communities in the body, including the lungs and skin. In particular, the solid medium approach to bacterial sample preparation adapts itself well to studying the effects of the mucus layers, important in many gastrointestinal and respiratory tract diseases. Building on previous results from other projects in the lab, we will use mutations and knockout strains to understand how bacteria behave in response to their environment, especially after treatment with antimicrobial drugs.

For informal enquiries: lindsay.baker@bioch.ox.ac.uk

Dr Lindsay Baker | Biochemistry (ox.ac.uk)

Project Code D2

Molecular mechanism of the MDM2-p53 mitotic timer pathway

Chromosome instability and aneuploidy delay progression through mitosis, triggering a p53 and p21 dependent cell cycle arrest in G1 thought to prevent proliferation of damaged cells ^1-6. This response is lost in cancer cells in which p53 has become inactivated by mutation or other mechanisms, including expression of viral oncoproteins ^7-10. Crucially, G1 cell cycle arrest following prolonged mitosis occurs even in the absence of detectable DNA damage, suggesting it has a different cause, proposed to be a direct consequence of the increased time spent in mitosis ^1-10.

We have recently discovered that MDM2, the p53 ubiquitin ligase, is a key component of the timer mechanism triggering G1 arrest in response to prolonged mitosis ¹¹. This timer function arises because MDM2 has a short half-life and ongoing protein synthesis is therefore necessary to maintain its steady-state concentration. Due to the attenuation of protein synthesis in mitosis, the amount of MDM2 gradually falls during mitosis, but normally remains above a critical threshold for p53 regulation at the onset of G1. When mitosis is extended by prolonged spindle assembly checkpoint activation, the amount of MDM2 drops below this threshold, stabilising p53. Subsequent p53-dependent p21 accumulation in the following G1 then channels cells into a prolonged cell cycle arrest, whereas abrogation of the response in p53-deficient cells allows them to bypass this crucial defence mechanism.

Ongoing aims of the project are focussed on understanding the molecular events in early G1 cells following delayed mitosis that result in exit from the cell cycle, and interplay between DNA damage signalling pathways and the mitotic timer in G1 and G2 cells.

References

1.        Wong, Y.L. et al. Cell biology. Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science 348, 1155-1160 (2015).

2.        Thompson, S.L. & Compton, D.A. Proliferation of aneuploid human cells is limited by a p53-dependent mechanism. J Cell Biol 188, 369-381 (2010).

3.        Li, R. & Zhu, J. Effects of aneuploidy on cell behaviour and function. Nat Rev Mol Cell Biol 23, 250-265 (2022).

4.        Thompson, S.L. & Compton, D.A. Examining the link between chromosomal instability and aneuploidy in human cells. J Cell Biol 180, 665-672 (2008).

5.        Uetake, Y. & Sluder, G. Prolonged prometaphase blocks daughter cell proliferation despite normal completion of mitosis. Curr Biol 20, 1666-1671 (2010).

6.        Yang, Z., Loncarek, J., Khodjakov, A. & Rieder, C.L. Extra centrosomes and/or chromosomes prolong mitosis in human cells. Nat Cell Biol 10, 748-751 (2008).

7.        Lambrus, B.G. et al. A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis. J Cell Biol 214, 143-153 (2016).

8.        Meitinger, F. et al. 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration. J Cell Biol 214, 155-166 (2016).

9.        Cuella-Martin, R. et al. 53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms. Mol Cell 64, 51-64 (2016).

10.      Fong, C.S. et al. 53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis. Elife 5 (2016).

11.      https://www.biorxiv.org/content/10.1101/2023.05.26.542398v2.full

Prof Francis Barr | Biochemistry (ox.ac.uk)

For informal enquiries: francis.barr@bioch.ox.ac.uk

Project Code: D3

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 D4

Nanomachines in the bacterial cell envelope

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.
• 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:

• Lauber et al. (2014) Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon. Nat Microbiol 9: 1089-1102.
• 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.

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 D5

Role of transposable elements in development to regulate gene expression

Transposable elements are mobile genetic elements that make up 50% of the mammalian genome. Transposable element mobility is dependent on their ability to produce a full-length transcript. This means, TE expression is not only dangerous due to transposition events, but also due to activation of regulatory sequences with the potential to influence gene expression. 

Because of the thread to genome integrity, cells have evolved a range of repressive mechanisms to prevent their expression. Epigenetic modifications such as DNA methylation and histone 3 lysine 9 trimethylation (H3K9me3) are particularly important. While older elements – more sequence diverse elements - have been linked to sequence-specific repression by Krüppel associated box (KRAB) zinc-finger proteins younger elements – with high sequence identify - tend to be silenced by the HUSH complex via H3K9me3. As a result, most TEs are transcriptionally silenced and not expressed in somatic cells. Interestingly, TE expression is essential for embryonic development. In mice, knock-down of specific TE families results in failure of embryos to progress past the 2-cell stage. Only 1-2% of TEs are young, full length, and still capable of transposition. However, evolutionarily young elements have highly similar sequences compared to older, more divergent TEs. Due to this, transcripts from young TEs cannot be mapped to their locus of origin with confidence using conventional short-read sequencing. Hence, detected expression could either be from a small proportion of young TE loci, or from all elements in a subfamily. We developed a long read RNA sequencing techniques called CELLO-seq to overcome these limitations. CELLO-seq enables us to map TE transcripts to their original genomic locus. As CELLO-seq also provides single-cell resolution, it allows us to determine TE expression heterogeneity between single cells in the embryo. Our work makes use of embryonic stem (ES) cell-based models to study molecular mechanisms by which Transposable elements influence gene expression. We use CRISPR/Cas9 based genome engineering to establish defined genetic backgrounds to test specific questions. We use next generation sequencing (NGS) and third generation sequencing (TGS) long read sequencing-based methods extensively, for example to map specific histone/DNA modifications and other chromatin features and to establish locus-specific transposable element expression. We make use of retrotransposition assays and flow cytometry based approaches to assess the role of transposon mobility in cells. We also use single molecule tracking approaches to investigate the dynamics of Transposable element RNA and its interaction with chromatin in early development. 

Dr Rebecca Berrens | Biochemistry (ox.ac.uk)

For informal enquiries: Rebecca.berrens@bioch.ox.ac.uk

Project Code D6

AI and MD to Understand Transporter Proteins  

This project will explore the use of a wide-range of computational techniques, ranging from QM/MM, atomistic MD, coarse-grain MD and AI models based on deep-learning to investigate how proton-coupled transporters work at the atomic level.  The work will be tightly coupled to ongoing structural biology work being undertaken in the Department.  Applicants should have a strong interest in computational chemistry and structural biology.

Prof Phil Biggin | Biochemistry (ox.ac.uk)

For informal enquiries: Philip.biggin@bioch.ox.ac.uk       

Project Code D7

Structural biology of DNA topology remodelling machines

Genetic information is stored in spectacularly large and thread-like molecules, the chromosomal DNA. This poses the fundamental “spaghetti problem”: DNA entanglements must be prevented by creating structured entities called nucleoids or chromosomes. Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases solve this issue in most if not all organisms.

We are interested in:

1. How ring-like SMC complexes use the power of ATP hydrolysis to extrude large DNA loops
2. How SMC complexes cooperate with DNA topoisomerases to prevent DNA entanglements
3. How pathogens such as bacteriophages and infective plasmids manipulate host chromosomes, and interact with host defences that use the mechanisms above

We address these questions using biochemical reconstitution, cryo-EM, and advanced bacterial genetics. We capture bacterial and viral genome remodelling machines in action, aiming to directly visualize how they work. Our long-term goal is a full mechanistic understanding of the processes that control the three-dimensional structure of chromosomes across the tree of life.

Dr Frank Bürmann | Biochemistry (ox.ac.uk)

For informal enquiries: frank.burmann@bioch.ox.ac.uk

Project Code D8

Single-molecule studies of chromatin replication

The copying of DNA, or DNA replication, is essential to cellular function. In eukaryotes it is carried out with high fidelity by a multi-protein complex called the replisome. Failure to do so can lead to defects in the copied genome, slow down or stall the replisome, and result in genomic instability or cell death. Mechanistic insight into normal replisome functioning is a prerequisite for understanding such failure and its downstream consequences.

This is an exciting time in eukaryotic DNA replication, as advances in genetics, biochemistry, and structural biology have identified the essential replisome components together with their stable arrangements. However, in operation the replisome is an active molecular machine with transient parts. Thus, a full mechanistic insight into this molecular machine requires a description of its dynamics. Because the replisome is active on DNA that is compacted into chromatin, this description must include the duplication and reassembly of all DNA-associated proteins. Understanding the coupling between these processes has fundamental implications for epigenetic inheritance and cancer.

Here, using an integrated approach at the interface of single-molecule biophysics and biochemistry, you will help to gain biophysical understanding of the operation of an individual eukaryotic replisome by examining its functioning on DNA and/or chromatin. In doing so, you will acquire a broad set of skills at the interface of single-molecule biophysics and biochemistry. You will start by first become familiar with the underlying molecular biology and biochemistry and learn to perform high-quality experiments in this context. You will then be introduced to state-of-the-art single-molecule techniques such as integrated force-fluorescence microscopy and TIRF microscopy. It is expected that using the combination of these biochemical and biophysical approaches you will gather data to examine several aspects of replisome functioning at the ensemble and single-molecule levels. To quantify the data that you will acquire, you will be introduced to and asked to make use of several forms of data analysis. The outcome of the project will contribute to new models of eukaryotic DNA replication that include a focus on its dynamic aspects.

Prof Nynke Dekker | Biochemistry (ox.ac.uk)

for informal enquiries: nynke.dekker@physics.ox.ac.uk

Project Code D9

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 D10

Chromosome associated mitotic inheritance and DNA methylation

Understanding how DNA methylation impacts chromosome structure and regulates epigenetic inheritance is important for understanding tissue specific gene regulation, genomic imprinting and X-inactivation. We will take a three-pronged approach to defining how 5-methylcytosine (5mC) and the factors that bind to this modification, influence cellular memory. Firstly, we will examine the physical and structural properties of metaphase chromosomes isolated from wildtype or DNA methylation-depleted (or degron-targeted) equivalents, using CryoET and optical tweezer approaches. This work will done in collaboration with Dr Lindsay Baker (Oxford Biochemistry) and Professor David Rueda (Imperial College London). We will quantitatively assess chromosome-associated proteins and RNAs that are enriched in each of these samples to identify candidate 5mC-dependent mitotic bookmarking factors. Finally, we will use this information to determine whether genomic imprinting, X-chromosome silencing or lineage-restricted gene expression, is perturbed by manipulating 5mC-dependent factors (either alone, or with other chromatin modifiers) during mitosis. This study will inform cell reprogramming strategies and may help in understanding how age-related changes in DNA methylation and histone H3K9me3 erode cellular memory.

References 

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

Djeghloul, D., Dimond, A., Cheriyamkunnel, S. et al. Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis. Nat Struct Mol Biol 30, 489–501 (2023). https://doi.org/10.1038/s41594-023-00943-7

Dimond, A., Gim, D.H., Ing-Simmons, E. et al. PBK/TOPK mediates Ikaros, Aiolos and CTCF displacement from mitotic chromosomes and alters chromatin accessibility at selected C2H2-zinc finger protein binding sites. (2024). https://doi.org/10.1101/2024.04.23.590758

Berger C, Premaraj N, Ravelli RBG, Knoops K, López-Iglesias C, Peters PJ.

Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.

Nat Methods 20, 499–511 (2023). doi: 10.1038/s41592-023-01783-5. PMID: 36914814

Meijering AEC, Sarlós K, Nielsen CF, Witt H, Harju J, Kerklingh E, Haasnoot GH, Bizard AH, Heller I, Broedersz CP, Liu Y, Peterman EJG, Hickson ID, Wuite GJL.

Nonlinear mechanics of human mitotic chromosomes.

Nature. 2022 May;605(7910):545-550. doi: 10.1038/s41586-022-04666-5. Epub 2022 May 4.PMID: 3550865

Prof Dame Amanda Fisher | Biochemistry (ox.ac.uk)

For informal enquiries: amanda.fisher@bioc.ox.ac.uk; 

Project Code D11

mRNA Therapeutics

mRNA vaccines have proven to be highly effective to combat infectious diseases. mRNAs can easily be designed and manufactured by in vitro transcription (IVT) using plasmid DNA as templates. IVT is a cheap and a simple, scalable chemical reaction that requires a plasmid DNA template, a simple DNA dependent RNA polymerase and ribonucleotides. The technology is highly versatile and cost effective and this together with the recent success of mRNAs vaccines has raised considerable interest to use mRNA technology for alternative applications including protein replacement, gene therapy and gene editing. The transition from mRNA vaccines to mRNA drugs that target non-infectious diseases however is challenging largely due to the complexity of targeting specific tissues and cells and the much higher protein threshold that are required for many of these alternative therapies. This project aims to address these issues by using and adapting our current mRNA therapeutic platforms to create new drugs.   

For informal enquiries: Andre.furger@bioch.ox.ac.uk

Associate Prof Andre Furger | Biochemistry (ox.ac.uk)

Project Code D12

The role of the centrosome in the spatiotemporal control of mitosis

General Background

In our laboratory we are fascinated by the molecular mechanisms that enable and control cell division. In particular, we want to understand how cells minimise chromosome mis-segregation events during mitosis to maintain their genome integrity. Proper chromosome segregation requires the formation of a functional spindle, composed of microtubules and associated proteins. Microtubules are focused in two spindle poles, organized by the two centrosomes.

Centrosomes are small cytoplasmic organelles that not only serve as mitotic spindle poles but also template cilia formation and contribute to signaling, trafficking, and organelle positioning. They are essential for normal cell proliferation; congenital mutations in centrosomal genes lead to various growth failure syndromes. Like DNA, centrosomes undergo semi-conservative duplication in S-phase, a process that ensures the presence of two functional centrosomes in cells when they enter mitosis.

Project Background

Aurora-A is a crucial protein kinase involved in centrosome maturation, mitotic entry, and mitotic spindle formation. Each role requires a specific binding partner, with interactions mediated by intrinsically disordered regions (IDRs)—protein domains that lack a stable three-dimensional structure. Despite their lack of defined structure, IDRs play important roles in various biological processes, including signaling pathways and transcriptional regulation, and are often mutated in diseases. Aurora-A has long been recognized as an oncogene, and its inhibitors are being explored for cancer treatments.

Recent results from our group suggest that centrosomes play a crucial signaling role by concentrating and activating Aurora-A kinase. In fact, the centrosome appears to be the only site in mitotic cells that can generate large amounts of T loop-phosphorylated (active) Aurora-A. The active kinase then forms a complex with TPX2 on the mitotic spindle and phosphorylates several important substrates. If Aurora-A skips centrosomal recruitment, it can still bind TPX2, albeit much less efficiently. Furthermore, the TPX2:Aurora-A complex, which uses non-phosphorylated Aurora-A, can still target some known substrates for phosphorylation but not all. This drop in kinase efficacy leads to spindle abnormalities and chromosome segregation errors.

Project Details

The primary aim of this project is to understand the rules governing Aurora-A's interactions with its substrates in cells.

Specifically, we aim to answer the following questions:

• Why do certain substrates, like the spindle pole protein NuMA, require Aurora-A activated at the centrosome, while others do not?
• When does centrosomal Aurora-A signal generation begin and end in cells, and can we modulate Aurora-A activity by altering this signalling window?
• How does Aurora-A (or the Aurora-A:TPX2 complex) differentiate between these substrates?
• Does the subcellular localization of the kinase and/or the substrates affect phosphorylation efficiency?
•  

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 D13

Mechanisms of ubiquitin signalling in DNA repair and disease

The DNA damage response functions as a cellular defense mechanism against DNA lesions that occur from either endogenous or exogenous sources. If left unrepaired, these DNA lesions pose a threat to cellular and organismal survival. To mitigate the potentially deleterious impact of DNA damage, eukaryotic cells have a highly connected network of DNA repair pathways that sense and repair DNA damage. How cells choose the correct DNA repair pathway is critical for their survival. Moreover, if there are multiple pathways available to cells, then they need to be able to balance the usage of these different pathways to control cell fate. One way that cells can direct specific DNA repair pathways is through the post-translational modification of target proteins involved in DNA repair. Ubiquitin (Ub) signaling is ideally suited to this task due its ability to form myriad polyUb chain topologies via different lysine (Lys; K) and methionine (Met; M) linkage types. More recently, the ubiquitin system has expanded even further, making it the perfect time to join the cutting-edge ubiquitin field (Lechtenberg and Komander (2024) Nature Structural & Molecular Biology).

Through this DPhil project, you will build on our recent unpublished findings and further our mechanistic understanding of the cellular roles that atypical and branched ubiquitin chains plays in the control of DNA repair pathway choice. To do this, you will be trained in a range of advanced cell biology approaches, including CRISPR-Cas9 genome editing, genetic screening, high-content single-cell microscopy, DNA replication fork analysis, DNA sequencing technologies, and ubiquitin proteomics. As well as deepening our understanding of these understudied ubiquitin chain types, the results of this research also will be essential to our on-going work to target parts of the ubiquitin system as potential therapies for cancer.

Dr Ian Gibbs-Seymour | Biochemistry (ox.ac.uk)

For informal enquiries from prospective students: Ian.gibbs-seymour@bioch.ox.ac.uk

Project Code D32

From bacteriocins to bacteriophages: structural biology of bacterial molecular machines

Bacteria thrive in different environments, adapting to the competition, predation, and fluctuating nutrients availability. Their diversity produces fascinating examples of protein machines with unique enzymatic activities.

Research in Ghilarov lab aims to reveal fundamental principles of how bacterial molecular machines use energy to manipulate three-dimensional structure of peptides and nucleic acids. Understanding these principles allows to control the activities of molecular machines, and ultimately to design artificial nanoscale devices. We are using a combination of cryoEM, X-ray crystallography, biochemistry, genetics and chemical biology approaches and collaborate with experts in modelling, protein design and single-molecule methods. Specific DPhil & MSc projects will be tuned to candidates’ interests, but must be related to one of the three lab themes:

Theme I: Ribosomally synthesized post-translationally modified peptides (RiPPs)

RiPPs enable bacterial competition, virulence, and defense against bacteriophages. RiPPs are genetically encoded, and converted into bioactive molecules by multienzyme complexes. Harnessing these complexes’ ability to modify custom peptides will allow synthesis of engineered molecules with programmed sequence. We are working on  different RiPP systems, including LAPs, lasso-peptides and thiopeptides, to get insights about RiPP biosynthesis, their ecological role, and to engineer them to create new-to-nature natural products[1,2].

Theme II: Bacterial DNA topoisomerases

Bacterial type II topoisomerases gyrase and topoisomerase IV are essential for removing positively supercoiled DNA in front of the progressing polymerases, introducing negative supercoiling required for chromosomal homeostasis, and segregating daughter chromosomes after replication. They work in close connection with the replisome and SMC proteins, directly and indirectly affecting all genomic transactions in the cell. The research in the lab aims to address a fundamental question of how topoisomerases use energy of ATP to introduce defined topology in DNA. We have developed cryoEM approaches to study gyrase and topoisomerase IV, resulting in a determination of the first gyrase structure in complex with chirally wrapped DNA[3]. We are also interested in protein and small molecule inhibitors of topoisomerases as inspiration for a new generation of antibacterial drugs[4,5], and in mechanisms of resistance to antibiotics related to topoisomerases.

Theme III: Bacterial antiviral defense systems

The field of bacterial phage immunity is rapidly expanding and is one of the most exciting topics of contemporary microbiology. We are studying a complex defense system called BREX (bacteriophage exclusion system). BREX components are dissimilar to any other proteins, and their functions cannot be easily predicted or deduced. Many of them bear similarity to eukaryotic proteins such as ORC/Cdc6. BREX complex assemble on DNA in a megadalton-size multiprotein complex, preventing phage replication by an unknown mechanism. We have recently determined structures of methytransferase BrxX[6] responsible for the self/non-self discrimination within the complex, and are actively investigating other components and homologous systems.

[1] Ghilarov et al. Mol Cell (2019) https://doi.org/10.1016/j.molcel.2018.11.032

[2] Travin et al. JACS (2018) https://doi.org/10.1021/jacs.8b02277

[3] Michalczyk et al. bioRxiv (2024) https://doi.org/10.1101/2024.04.12.589215

[4] Bakker et al. Nat Chem (2024) https://doi.org/10.1038/s41557-024-01516-x

[5] Michalczyk et al. Nat Catal (2023) https://doi.org/10.1038/s41929-022-00904-1

[6] Drobiazko et al. bioRxiv (2024) https://doi.org/10.1101/2024.04.12.589305

Ghilarov lab website: ghilarovlab.com

Contact email address for informal enquiries from prospective students: Dmitry.ghilarov@jic.ac.uk

Project Code D14

Structural biology of host-parasite interactions and structure-guided vaccine design.

Prospective applicants can learn about our research programs from our website (higginslab.web.ox.ac.uk) and we have a variety of possible projects related to host-parasite interactions and structure-guided vaccine design. Contact Matt for more information.

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 D15

Understanding mechanisms underlying chromatin-based inheritance

Our lab aims to discover how chromosome structure and patterns of gene expression are maintained through mitotic and sometimes even meiotic divisions.

How components other than primary DNA sequence, such as proteins, that are part of chromatin and govern gene activities and chromosome structure are maintained and replicated through cell division is not understood. We are interested in resolving this.

The Human Centromere

Centromeres are chromosomal loci that form the attachment site for spindle microtubules, driving chromosome segregation in mitosis. Centromeres are defined by a unique chromatin structure featuring the histone H3 variant CENP-A. Nucleosomes containing CENP-A are at the centre of an epigenetic feedback loop where chromatin-bound CENP-A is sufficient to initiate self-templated inheritance of centromeres, even on ectopic genomic loci, called neocentromeres. A DPhil project will be focussed on questions related to how CENP-A chromatin domains form de novo, how CENP-A chromatin can be transmitted through cell division and how its assembly is cell cycle controlled.

Key methodologies include mammalian cell culture, microscopy-based fluorescent pulse labelling technologies to visualize both ancestral as well as nascent pools of CENP-A histones, live cell imaging, chromatin immunoprecipitation (ChIP), CUT&RUN-seq, as well as the latest nanopore long-read sequencing methods to understand chromatin structure at complex repeats, as well as CRISPR-based genome editing to establish cell biological tools.

We run a dynamic team of postdocs and students that offer expertise and support. See www.jansenlab.org for more information. Do get in touch if you have any queries.

Prof Lars Jansen | Biochemistry (ox.ac.uk)

For informal enquiries: lars.jansen@bioch.ox.ac.uk

Project Code D16

Tackling antimicrobial resistance using molecular dynamics and artificial intelligence

1. 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.

2. 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 D17

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: D18

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

1. Venugopal, K. et al. Nat. Rev. Microbiol. 2020 183 18, 177–189 (2020). https://doi.org/10.1038/s41579-019-0306-2
2. Schrevel, J. et al. Parasitology 135, 1–12 (2008). https://doi.org/10.1017/S0031182007003629
3. Fowler, R. E. et al. Parasitology 117, 425–433 (1998). https://doi.org/10.1017/S003118209800328X
4. 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 D19

Toll signalling in Drosophila intestinal regeneration

Brief description of project(s) or research theme (no more than 500 words): The gut is an interphase where epithelial renewal and microbiota maintenance are balanced with immunological regulation. How these functions integrate with cellular signalling is unclear. In the Drosophila gut, asymmetric mitosis of Intestinal Stem Cells (ISCs) produces Enteroblasts (EBs) that differentiate to Enterocytes (ECs). By using cell-specific RNAi we found that the immune receptor Toll, is needed for ISC mitosis in homeostasis and infection. Conversely, Toll gain of function in ISCs and EBs pushed ISCs into mitosis to produce EBs but blocked EB to EC differentiation, resulting in intestinal dysplasia. Toll activity was mediated by the classical Toll pathway components, JNK and Akt/mTOR signalling. These phenotypes were independent of the microbiota as seen in germ free flies. However, Toll activation resulted in increased density of commensal gut bacteria by transcriptional suppression of host lysozymes and amidases. These results indicate Toll as a major evolutionary conserved regulator of the intestinal landscape. The project will extend on these results and delve deeper on the metabolic requirement of ISC mitosis as well as the role of microbiota as a means to prevent the establishment of an enteric pathogen.

Prof Petros Ligoxygakis | Biochemistry

For informal enquiries: petros.ligoxygakis@bioch.ox.ac.uk

Project Code D20

“Deconstructing human ribosomal-RNA processing by Nanopore sequencing and TREX”

Introduction:

Ribosomes are universally conserved, essential molecular complexes responsible for protein synthesis in all living organisms. The mature human ribosome (80S) consists of four ribosomal RNA (rRNA) molecules—18S, 5.8S, 28S, and 5S rRNAs—alongside 80 ribosomal proteins (RPs), which together form the small  and large subunits of the ribosome. Ribosome synthesis begins in the nucleolus with the transcription of a large polycistronic pre-rRNA transcript that includes 18S, 5.8S, and 28S rRNA sequences, interspersed with four spacer segments (5'ETS, ITS1, ITS2, 3'ETS)¹. These spacer sequences undergo cleavage and degradation during a complex set of step-by-step processing events. While the responsible factors and the sequences of pre-rRNA processing events are well understood in yeast, significant gaps remain in our understanding of pre-rRNA processing in higher eukaryotes such as humans¹.

Objectives:

This project aims to tackle the above problem by elucidating the mechanisms of human pre-rRNA processing through the use of two innovative approaches: Nanopore Direct RNA Sequencing (DRS)², and Targeted RNase H-mediated Extraction of X-linked RBPs (TREX)³.  Specifically, our project consists of three major aims:

1: Using Nanopore DRS of pre-RNAs, we aim to unbiasedly identify and quantify the different pre-rRNA transcript intermediates that arise during rRNA processing, and define the order of their appearance and removal. Importantly, deciphering the detailed steps of rRNA processing requires the concurrent analysis of 5′ and 3′ ends, a feature that can only be revealed through long-read sequencing technologies such as Nanopore². We also aim to reveal novel routes of pre-rRNA processing that may be triggered by various internal and external cues, such as specific signal transduction pathways, or cellular stresses like hypoxia.  

2: Utilising TREX, a new powerful method developed by our group that allows unbiased mapping of protein-RNA interactions³, we aim to map the protein binding partners of each crucial pre-rRNA segment (e.g. sites of cleavage), identifying known and novel rRNA processing factors in human ribosome biogenesis.

3: Through use of RNA interference (RNAi) and CRISPR-Cas9, we will then test the functional significance of any newly pre-rRNA binding factors identified by TREX, in mediating specific pre-rRNA processing steps. This will be achieved by selective depletion of the candidate factors, combined with Nanopore DRS of pre-rRNA transcripts, to assess accumulation or loss of specific pre-rRNA species. Additionally, we will assess whether the different signalling pathways or stress conditions change the functional impacts of the identified factors. Finally, using purified proteins and synthetic matching pre-rRNA sequences, we will validate the functional roles of these factors in vitro, at the molecular level.

Outcomes:

Together, this project aims to resolve a key outstanding question in the ribosome field: the precise order and mechanisms of pre-rRNA processing in higher eukaryotes. In addition, we hope to reveal the dynamics of this process in response to various signalling cues and cellular stresses. Understanding these mechanisms will provide critical insights into ribosome biogenesis and its regulation in higher eukaryotes, with potential implications for understanding diseases linked to ribosomal dysfunction.

References:

1   Dorner, K., Ruggeri, C., Zemp, I. & Kutay, U. Ribosome biogenesis factors-from names to functions. The EMBO journal 42, e112699, doi:10.15252/embj.2022112699 (2023).

2   Grunberger, F., Juttner, M., Knuppel, R., Ferreira-Cerca, S. & Grohmann, D. Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea. RNA 29, 1255-1273, doi:10.1261/rna.079636.123 (2023).

3   Dodel, M. et al. TREX reveals proteins that bind to specific RNA regions in living cells. Nat Methods 21, 423-434, doi:10.1038/s41592-024-02181-1 (2024).

For informal enquiries: faraz.mardakheh@bioch.ox.ac.uk

Project Code D21

Understanding the role of ion channels in Parkinson’s Disease and the link to lysosomal homeostasis.

Lysosomes are traditionally characterized as the terminal recycling centre in cells due to their unique acidic properties. However, recent studies have identified lysosomes as playing critical roles in regulating metabolism and inflammation. Key to these functions are the integral membrane proteins, both channels and transporters, that exist in the lysosomal membrane and control the influx and efflux of ions and metabolites and are essential for maintaining the acidic pH environment. Transmembrane protein 175 (TMEM175) is a unique lysosomal potassium channel that shares little sequence or structural similarity with other potassium channels. Genome-wide association studies in humans and functional studies in mouse models have established that certain variants in TMEM175 act as a genetic risk factor in the common form of Parkinson's disease (PD). Subsequent studies have identified TMEM175 activators as potential therapeutics. However, the molecular mechanism of TMEM175 function and its role in PD progression remain unclear. This project will focus on addressing these key questions. Specifically, the project will involve developing novel nanobody binders to understand the molecular basis for TMEM175 activation and interaction partners in the lysosomal membrane. The student will be trained in cutting edge structural biology methods, including cryo-EM, biophysical methods, and electrophysiology. 

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

Targeting DNA repair mechanisms in precision cancer therapies

Cancers often have defects in processes that repair damage to their genetic material and tumours survive by becoming ‘addicted’ to processes that compensate for loss of these mechanisms. This is an Achilles heel of the tumour and using drugs to interfere with these pathways kills cancer cells. Principal in this strategy is using small molecule inhibitors of PARPs, a family of enzymes that modify proteins with ADP-ribose to promote DNA repair. PARPs are critical for the survival of cells with defects in the BRCA1 and BRCA2 breast and ovarian cancer genes and PARP inhibitors are used to kill these tumours.

However, despite the success of PARP inhibitors in the clinic, the effectiveness of PARP inhibitors differs between patients and tumours develop resistance to this treatment. The mechanistic basis of how PARPs regulate DNA repair is unclear, representing a fundamental barrier to addressing these challenges. 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 and MSc projects are available that integrate cutting edge genome engineering, proteomics and cell biology to address the following:

a) How do PARPs become activated in response to DNA damage?

b) What proteins do they modify at sites of DNA damage?

c) How do these modifications regulate DNA repair?

These multidisciplinary hypothesis-driven research projects will increase our understanding of how PARPs regulate DNA repair and provide critical information to develop novel strategies that target PARPs to treat a variety of pathologies

Prof Nick Lakin | Biochemistry (ox.ac.uk)

For informal enquiries: nicholas.lakin@bioch.ox.ac.uk

Project Code D23

Mechanisms of Wnt signalosome formation and maintenance.

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 (Rim et al 2022). 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. The aim of this DPhil project is to use biochemical and biophysical methodologies to dissect the steps leading to the Wnt signalosome formation and maintenance. This will be through a combination of both in vitro protein biochemistry and cell-based approaches. This project complements other projects within the lab and thus provides ample opportunities to collaborate within the lab.

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.

References:

1. Ranes, M., Zaleska, M., Sakalas, S., Knight, R. & Guettler, S. Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture, phosphorylation, and ubiquitylation. Mol Cell 81, 3246-3261.e11 (2021).

2. 1.Rim, E. Y., Clevers, H. & Nusse, R. The Wnt Pathway: From Signaling Mechanisms to Synthetic Modulators. Annu. Rev. Biochem. 91, 571–598 (2022).

Dr Michael Ranes | Biochemistry (ox.ac.uk)

For informal enquiries: michael.ranes@bioch.ox.ac.uk

Project Code D24

Evolution of small non-coding RNA pathways

Small non-coding RNAs are central regulators of eukaryotic gene expression, and appear across eukaryotic organisms, suggesting an ancient origin in the last common ancestor of all eukaryotes.  However, small non-coding RNA pathways and mechanisms evolve extremely rapidly, such that many pathways have been lost independently in different lineages.  One standout example are Piwi interacting small RNAs (piRNAs).  piRNAs are ancestral to animals and highly conserved such that similar piRNA biogenesis mechanisms are found in organisms as diverse as molluscs, fruitflies and humans.  They have crucial roles in controlling transposable elements, and loss of piRNAs leads to loss of fertility, often linked to rampant transposon reactivation.  However, piRNAs have been lost a number of times independently, most strikingly in nematodes.  Whilst the well-characterised model nematode Caenorhabditis elegans has a piRNA pathway at least 5 independent lineages have lost piRNAs completely.  So far little is known about the factors that predispose nematodes to lose piRNAs, or the forces that drive loss of the piRNA pathway.  In this project we aim to uncover more about what might be responsible for the loss of the piRNA pathway. We will use comparative genomics taking advantage of the extensive database of nematode genomes to identify genomic features that co-evolve with piRNA presence/absence.  We will also take advantage of a close relative of C. elegans that has lost piRNAs (Caenorhabditis plicata) to experimentally test key hypotheses about why these genomic features are associated with piRNA evolution.  Together,  this joint computational and wet lab approach will deliver new insights into why piRNA evolution is so rapid, and give new insights into the most important conserved functions of piRNAs across animals.  It will offer training in a range of computational methods, including comparative genomics, high-throughput sequence data analysis and machine learning to identify correlations and test the strength of associations.  It will also provide training in wet-lab experimental techniques including nematode culture and laboratory evolution of nematodes. 

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 D25

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 D26

Protein structure and interactions in health and disease .

Our laboratory seeks to understand how protein functions arise from their molecular structure and interactions, and how these processes are involved in human health and disease. Projects in at least two areas are possible. (1) We have a long-standing interest in the molecular mechanisms by which Influenza virus proteins function. 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) More recently, we have begun to study the structure and function of chaperone proteins, especially J-domain proteins, to understand how they interact with misfolded/unfolded clients and with the HSP70 machinery.

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 D27

Structural biology of brain development

How does a single cell give rise to the amazingly complex human brain?

Brain development hinges on the proliferation and self-assembly of cells to form highly organized brain tissues and circuits. This intricate process relies on specialized receptor proteins that orchestrate the movements of brain cells during development. These receptors, situated on the cell surface, function like a cellular navigation system: they detect various cues (ligands) in the cellular environment and initiate signaling cascades that guide cells in the right direction. Collectively, these receptor-ligand interactions ensure the correct positioning of cells and their axons/dendrites within brain tissue, thereby coordinating the formation of brain circuits.

Approach

Our lab is part of a network that adopts an interdisciplinary approach to understand molecular functions within complex tissues. We specialize in structural biology (especially cryo-electron microscopy and X-ray crystallography) and advanced cell biology techniques. We integrate our findings with cryo-electron tomography, in vivo models, and super-resolution fluorescence microscopy through close collaboration with world-leading experts in Oxford (Baker), Barcelona (del Toro), and Bordeaux (Naegerl).

Outcome

By leveraging our established experimental pipeline, you will lead an interdisciplinary project aimed at unraveling how specific receptor-ligand interactions direct brain development. This project will yield groundbreaking discoveries about the molecular mechanisms underlying brain formation and function, much of which remains unknown. The findings will be directly relevant to brain disorders associated with these receptors. The project offers training in multiple techniques, particularly in structural biology and cell biology. Additionally, you will benefit from regular meetings with collaborators, providing further training and networking opportunities. Our team frequently engages in hands-on collaborative work in other labs within MIGRATE: 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 D33

Virus Glycosylation and Molecular Mechanisms of Host Recognition

We are interested in understanding the role of complex carbohydrates, or glycans, in viral infection, specifically uncovering fundamental aspects of how enveloped viruses modify their surface proteins with sugars and how this relates to host attachment via cell surface glycosaminoglycans, neutralising antibodies and innate immune lectins. Our work focuses on HIV, Ebola, Lassa and coronaviruses and is founded on the development of two advanced structural and biophysical techniques – mass spectrometry and mass photometry.

This interdisciplinary project aims to characterise how viruses glycosylate their surface proteins (e.g. HIV-1 Env or Ebola GP) and use this information to improve the design of structure-based vaccines. Importantly, we have found that glycosylation of viral glycoproteins isolated from infectious virions differs to isolated glycoproteins made in the lab, which implicates the role of cellular glycosylation machinery and/or structural dynamics of the underlying protein. This observation is important for the design of vaccines as well as our basic understanding of enveloped virus structural biology in general. The aim of this project will not only explore how viral proteins are glycosylated but also how changes affect interactions with host biomolecules, including antibodies and cell surface glycosaminoglycans (GAGs) using single-molecule imaging.

This work will entail an integrated approach using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and mass photometry (MP) - you will have the opportunity to be at the cutting-edge of developing several methods in MS and MP. HDX-MS is a sensitive structural biology technique capable of probing the conformational dynamics of proteins in solution, providing information of binding site location and structural changes of target proteins upon antibody or GAG binding (i.e. tracking binding-associated allostery). MP is a single molecule light scattering-based technology that we have developed which enables detailed quantitative information of binding stoichiometry and affinity. The integration of both HDX and MP is unique and in doing so we will expand our understanding of the molecular determinants of the antibody-mediated immune response and cell attachment by enveloped viruses

For more information about the Struwe lab see; https://struwe.web.ox.ac.uk

Associate Prof Weston Struwe | Biochemistry (ox.ac.uk)

For informal enquiries from prospective students: weston.struwe@bioch.ox.ac.uk

Project Code D28

Bacterial infection, stress response, DNA repair, antibiotic resistance

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

Understanding molecular mechanism that transcription of the non-coding genome  

Recent technological advances have revealed a plethora of diverse long non-coding (nc) RNA molecules produced from eukaryotic genomes. Mutations in non-coding regions of the genome and altered expression of ncRNAs underpins a number of pathologies including cancer. Yet, very little is known about mechanisms involved in production of ncRNAs preventing us from understanding their role in health and disease. Our previous work lead to discovery that in contrast to mRNAs, nc transcripts rely on distinct and poorly understood mechanisms that control their RNA polymerase II (Pol II) transcription. As a result, ncRNAs are non-polyadenylated and targeted by the cellular RNA degradation machinery, RNA exosome.

The PhD project aims to fill the key gaps in our understanding of the transcriptional mechanisms involved in regulation of ncRNA. This will be achieved through the Aims 1-3. A PhD student will identify and characterise transcription complexes linked to production of ncRNA biochemically (Aim 1) and investigate how these complexes are recruited to Pol II during transcription and how they control biogenesis of ncRNA in human cells using state-of-the-art genomic approaches (Aim 2 and 3).

Associate Prof Lidia Vasilieva | Biochemistry (ox.ac.uk)

For informal enquiries: Lidia.vasilieva@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

Project Code D31

Molecular mechanisms underlying viral evolution and host changes

We seek to understand how enveloped viruses evolve and use this knowledge to come up with ways to deal with future pandemics.

Many deadly human pathogens, such as influenza and SARS viruses, are made up of just a few components but can infect a number of different hosts. How is it possible that these components suffice to fulfil all the functions necessary for a virus to infect the cell and then to assemble into a new viral particle? How do viral proteins perform multiple functions and how does the virus manage to retain all these functions as it evolves? How does a virus infect different hosts using the same set of its own proteins to engage a range of machineries of different hosts? And, finally, how does a virus evolve and ‘learn’ to optimise its interactions with a new host?

The recent COVID-19 pandemic has demonstrated that these questions are key to understand where new viruses come from and how they evolve upon transmitting to a new host. We have used biochemical, biophysical, and structural methodologies, mainly cryoEM, to reveal the mechanisms by which SARS-CoV-2 became able to infect humans and then further evolved to optimise viral infectivity in the variants of concern. [1-7]

The lab continues to work on coronaviruses and studies influenza viruses to understand how their proteins achieve the versatility needed to infect diverse hosts and fulfil multiple functions during infection. In particular, we want to explain how related viral strains use similar glycoproteins to engage receptors as different as glycans and proteins. We are also interested in understanding how viral proteins come together during virus assembly: how do they find other viral components, how do they ‘coerce’ the host to transport them, and how do they exclude hosts proteins from growing viral particles.

Tackling these questions can directly impact public health. The more we understand the rules governing the evolution of viral proteins, the better we can predict the impact of emerging viruses and thus increase our pandemic preparedness. Our long-term aim is to use structural and mechanistic insights to guide design of much-needed new antivirals and vaccines against zoonotic viruses.

Several projects encompassing areas above are available in the lab. If you are interested, please apply and do not hesitate to get in touch earlier to discuss more details. I am committed to working with you to scope projects that combine your interests in specific research questions as well as methodologies, your long-term career aspirations, and available expertise and pilot data in the lab. In this way we will devise the MSc or DPhil project that bests suit you.

REFERENCES (#co-first, •corresponding):

1. Antoni G. Wrobel# (2023) “Mechanism and evolution of human ACE2 binding by SARS-CoV-2 spike” Current Opinions in Structural Biology 102619.

2. Valeria Calvaresi#, Antoni G. Wrobel#, Joanna Toporowska, Dietmar Hammerschmid, Katie J. Doores, Richard T. Bradshaw, Ricardo B. Parsons, Donald J. Benton, Chloë Roustan, Eamonn Reading, Michael H. Malim, Steven J. Gamblin, Argyris Politis#. (2023) “Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein” Nature Communications, 14 (1), 427

3. Antoni G. Wrobel*#, Donald J. Benton*#, Chloë Roustan, Annabel Borg, Saira Hussain, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin# (2022). “Evolution of the SARS-CoV-2 spike in the human host” Nature Communications 13, 1178.

4. Antoni G. Wrobel*#, Donald J. Benton*#, Pengqi Xu, Annabel Borg, Chloë Roustan, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin# (2021). “Structure and binding properties of Pangolin-CoV Spike glycoprotein inform the evolution of SARS-CoV-2.” Nature Communications, 12 (1), 837.

5. Donald J. Benton*#, Antoni G. Wrobel*#, Chloë Roustan, Annabel Borg, Pengqi Xu, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin#. (2021) “The effect of the D614G substitution on the structure of the spike glycoprotein of SARS-CoV-2” Proceedings of the National Academy of Sciences, 118(9), e2022586118.

6. Donald J. Benton*#, Antoni G. Wrobel*#, Pengqi Xu, Chloë Roustan, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin# (2020) “Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion” Nature 588(7837), 327–330.

7. Antoni G. Wrobel*#, Donald J. Benton*#, Pengqi Xu, Chloë Roustan, Stephen R. Martin, Peter B. Rosenthal, John J. Skehel, Steven J. Gamblin#  (2020) “SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects.” Nature Structural and Molecular Biology 27, 763–767 (2020).

for informal enquiries:

Current: antoni.wrobel@crick.ac.uk

From 01/09/24: antoni.wrobel@bioch.ox.ac.uk