Genome duplication is essential for cell proliferation. Errors in the mechanisms that control DNA replication can cause genomic instability and lead to the development of genetic diseases and cancer. In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication. How changes in protein structure regulates function during key events in origin activation and replisome progression, such as melting of the DNA duplex upon CMG helicase assembly, or the mode of binding of Pol alpha during primer synthesis remain unknown. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand DNA unwinding and replisome architecture. To truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. To address this issue, I will integrate single-particle cryo electron-microscopy (cryo-EM) with sophisticated biochemical approaches to image origin-dependent DNA replication reactions in vitro on native DNA substrates. To do so, I will establish short origin-containing DNA substrates that permit loading of a single bidirectional replication fork, allowing large numbers of protein bound origins to be visualised within a single field of view. Initially, I will investigate how the structure of duplex DNA changes upon CMG formation during origin activation. Next, I will examine the molecular mechanisms of primer synthesis after origin activation using both cryo-EM and in vitro DNA replication reactions. Finally, I will capture and image synthesising intact replisomes at near atomic resolution. By visualising entire DNA replication reactions instead of isolated replication complexes at high resolution, we will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication.

Myocardin-related transcription factors (MRTFs) are G-actin binding proteins which act as transcriptional co-activators Myocardin-related transcription factors (MRTFs) are G-actin binding proteins which act as transcriptional co-activators in association with the transcription factor SRF. MRTF-SRF target genes encode numerous proteins involved in actin dynamics, cell adhesion, migration and contractility. The subcellular localization of the MRTFs is controlled by actin binding which inhibits their nuclear import and promotes their nuclear export. Extracellular signals which activate Rho-family GTPases induce actin polymerization and G-actin depletion, which induces MRTF shuttling to the nucleus and transcriptional activation of MRTF-SRF target genes. The MRTF-SRF pathway activation via Rho plays an important role in cancer cell invasion and metastasis. In addition, in MRTF-SRF signalling inhibits cell senescence in hepatocarcinoma cells which present high Rho activity, but this has not been investigated in other cell types or cancer models. Cell senescence is a process of cell-cycle arrest which typically occurs in ageing, cancer, development or tissue repair, and can facilitate recruitment of immune cells. In the tumour microenvironment, senescent cells can direct events such as therapeutic resistance or metastasis that support malignant progression. In this project we will determine the molecular mechanisms by which MRTF-SRF signalling inhibits cell senescence in MEFs. We also aim to investigate the possibility for it to modulate melanoma progression in the BRafV600E mouse model, where senescence is an initial step prior tumour transformation. Increasing evidence suggests that anti- and pro-senescent therapies can be beneficial also in other pathologies, such as fibrosis, by limiting cell proliferation and allowing clearance of damaged cells. These studies have the potential to reveal new approaches to the modulation of senescence pathways for therapeutic benefit.

Inflammation is a host response that evolved to counteract noxious stimuli that result from infection or tissue injury, and serves to return the affected tissue to homeostasis. Cell death-associated sterile inflammation is a major contributor to secondary tissue damage associated with multiple conditions such as myocardial infarction, transplantation, and stroke. Damaged tissues are thought to elicit their inflammatory effects through the sudden release from cells of endogenous Damage-Associated Molecular patterns (DAMPs) that serve to recruit and modulate the function of immune cells. In vertebrates, a diversity of molecules have been implicated as DAMPs, including ATP, uric acid, and F-actin. In mammals, F-actin is recognised as a DAMP by the C-type lectin receptor DNGR1, expressed on CD8+ Dendritic cells (DCs), that signals to favour the cross-presentation of dead-cell antigen to CD8+ T-cells. Independently of its work on DNGR-1, the host laboratory discovered that extracellular actin elicits a JAK-STAT-dependent inflammatory response in the fruit fly (Drosophila melanogaster). DNGR-1 does not have a functional homolog in fly, therefore the actin sensor remains obscure. In order to identify the molecular sensor of extracellular actin we have conducted an in silico-based screen to identify a candidate list of potential sensors. To functionally evaluate these candidates, we will conduct in vivo RNAi and in vitro gain-of-function screens in Drosophila. We will validate the role for this novel sensor in mediating sensing of extracellular actin through multiple genetic and biochemical approaches. We expect our proposal to give novel insights into the signalling transduction and immunobiology of host responses to evolutionary conserved DAMPs. We anticipate that by understanding cytoskeletal-mediated innate inflammatory responses in fly, it will provide important insights into the evolution of similar damage sensor response pathways in higher organisms

The immense variety of behaviours displayed by animals – including ourselves- arose through the evolution of neural circuits within the brain. However, how central neuronal circuits change over time, integrating these modifications within the brain's highly interconnected networks remains largely unknown. Particularly puzzling is that evolution acts at the level of the genome but behaviour arises through the activity of neuronal networks. How are these two levels integrated? How do evolutionary changes on the genome lead to changes in neuronal circuits that themselves lead to behavioural divergences across animals? The larval olfactory system of Drosophila provides a powerful model to address these questions because: 1) different Drosophila species have evolved diverse odour-guided behaviours, 2) the olfactory system is numerically simple yet parallels more complex circuits, and 3) the availability of molecular tools and resources. I will use this system to identify hotspots of central neuronal circuit evolution at both functional and genetic levels by comparing two Drosophila species. First, leveraging comparative connectomic data from the host lab, I will use calcium imaging and modelling to examine the functional impact of cross-species connectomic changes. Second, I will employ a combination of transcriptomic approaches to determine the molecular changes that lead to functional and connectivity differences. Finally, I will apply these insights to re-engineer the neuronal circuits across species by making use of the genetic tools available in D. melanogaster. This research will enhance our understanding of the intricate interactions between genes, neuronal circuits, and behaviour in the context of evolution.