DNA double-strand breaks (DSBs) are a highly toxic form of DNA damage that can kill cells and cause the types of mutations to the DNA in our cells that can trigger cancer. This cell-killing property of DSBs explains why they are intentionally generated in radiotherapy and chemotherapy treatments to eliminate cancer cells. However, DSBs also arise spontaneously in normal dividing cells, when the cell's DNA copying machinery encounters problems. These DSBs are typically recognised and processed by error-free DNA repair pathways, preventing them from causing the mutations that trigger cancer development. Perhaps paradoxically, in certain specialised tissues mutagenic DSB repair is actually favoured, providing a molecular mechanism by which genetic material can be transferred between different regions of the genomes in our cells to create genetic diversity. These mutagenic events are fundamental for the plasticity of our adaptive immune systems, reshuffling antibody gene segments in white blood cells so that they can generate the many different types of antibody that are needed to fight the many different pathogens that we are exposed to. To counteract the potentially hazardous effect of unscheduled DSBs and respond appropriately when they are programmed, cells have evolved two dedicated DNA repair pathways: Homologous Recombination (HR) uses a copy and paste mechanism to repair DSBs accurately. It is therefore the most important pathway in dividing cells, as this is when most DSBs occur and the threat of new cells inheriting new mutations means accurate DNA repair is mandatory; On the other hand, Non-homologous end-joining (NHEJ) is a more simple pathway that can glue nearly any two DNA ends together, not caring about their sequence or whether they are the right ones. NHEJ normally repairs DSBs in non-dividing cells where they occur as isolated events, yet when numerous DNA ends are available it can be mutagenic. NHEJ is also crucial in our immune systems, where the fusing of DNA ends originating from two DSBs generated at different locations on our chromosomes is the required outcome. Because of this intrinsic discrepancy in desired DNA repair outcome between different cellular contexts, achieving the right equilibrium between these repair pathways is vital to ensure DSBs are appropriately resolved. Recently, we have found imbalances in DNA repair pathway equilibrium to link normal immune-function to human breast cancer development. Indeed a specialised subset of proteins that normally facilitate antibody gene rearrangements, are also responsible for the mutations that accompany loss of the human breast cancer tumour suppressor 'BRCA1', driving malignant transformation. In new work, we have identified a new protein whose loss in BRCA1-deficient cells can rescue the DNA repair defect that normally afflicts them. However, this then renders them resistant to important anti-cancer drugs that are normally extremely effective at killing them. Importantly, we have found that this protein is likely to be crucial for normal immune function, promoting DNA repair in antibody genes. Our proposal extends on these exciting findings, using molecular, biochemical, and cell biology approaches to question the molecular basis of this protein's unanticipated function in DSB repair. We will also inactivate this gene in the mouse, to study its actual role in the immune system, and generate important reagents for in vitro studies. The benefit of this work will be two-fold: Firstly, it will provide new insight into normal immune function, aiding the understanding of human immunodeficiency disorders; Secondly, it will reveal the molecular basis of common human cancers, also yielding insight into potential mechanisms of drug resistance that face personalised medicine approaches. These findings may pave way to improved approaches to diagnose, treat and better manage cancer.

Malaria is caused by a parasite that is transmitted exclusively by mosquitoes. The greatest malaria reduction and eradication success stories have been achieved by interrupting transmission. Historically, this has been through mosquito control. Targeting the small population of specialised parasites residing in the human host that are transmitted through mosquitoes would provide a similarly powerful malaria control method but we know too little about this population. Until now, the genes expressed by parasites have been analyzed by combining millions of parasites together. This approach confounds differences between individual parasites that could underlie success in getting into another host or in resisting the drugs we use to kill the parasites. We have recently developed a method to analyse single parasites one at a time. This technological leap has allowed us to understand parasites in the laboratory with more precision than ever before and importantly to understand how one parasite may differ from another during the whole life of the parasite both in the host and in the mosquito. Although the laboratory setting and lab strains of parasites are powerful tools for understanding parasite biology, in the lab we cannot understand the full diversity of parasites that exist in the wild causing devastating consequences for infected individuals. In this project we propose to characterise wild parasites at an individual level in partnership with Malian scientists. Our exploration will allow us to characterise the three main species of malaria parasite in sub-saharan Africa on a single parasite level for the first time. We will integrate the data into an interactive website called the Malaria Cell Atlas. This will become a key resource for the research community. We will then explore the changes from one patient to the other of the deadliest malaria species in both patients that are suffering from malaria symptoms and also from infected carriers who are not suffering from malaria, both of which contribute to the overall reservoir of parasites. Altogether, we will look at more than 300,000 individual parasites and get a very deep understanding of how individual parasites are both similar and different from each other. Understanding this infectious reservoir is pivotal to identifying how parasites efficiently get from one person to the next. Altogether our findings using cutting-edge tools to explore wild parasites will be key to understanding malaria and how to best control it.

We propose to provide state of the art analysis and annotation of the pig genome sequence being generated by the International Pig Genome Sequencing Project. We will make the annotated genome sequence accessible on the Web through the Ensembl site at http://www.ensembl.org . The pig genome is the entire DNA sequence of the pig which defines all the biological molecules that make up a pig. By acquiring, managing and annotating the pig genome sequence one accelerates research for both pig biology and for mammalian biology. Impact on pig biology: Because of the extensive selective breeding which has occurred during domestication, there are a considerable number of breed or line-specific features, from fat/muscle ratios, litter size to skin colour. These features can be mapped genetically into broad regions of the genome, but the final identification of the genes responsible and the causal genetic variation is very complex. The availability of a well-annotated pig genome sequence with links to other data sources, especially those on phenotypes such as growth, carcass composition or responses to infectious disease would provide a dramatic boost to the identification of these causative genes.

Childhood diarrhoea and bacterial bloodstream infections account for a considerable proportion of illnesses and deaths among children under five years of age worldwide. The under-five mortality produced by these infections is disproportionately high in Nigeria and other parts of Africa. This study proposes to examine two causes of these infections, enteroaggregative Escherichia coli and Salmonella, and to identify bacterial lineages that account for a significant proportion of childhood diarrhoeas and invasive infections among Nigerian children. Stool specimens will be obtained from children with diarrhoea and from healthy children attending clinics in Ibadan, Nigeria. E. coli and Salmonella will be isolated from the specimens and characterized at the molecular level to identify disease-causing strains and the disease-causing genes these subtypes carry. Enteroaggregative Escherichia coli and Salmonella isolates will be subjected to further analysis, involving sequencing parts of their genomes. The resulting DNA sequences will be compared to determine inter-relationships among different genetic lineages of bacteria isolated in this study and between these isolates from Nigeria and strains other parts of the world. These analyses will reveal how disease-causing lineages change over time and are transmitted locally and globally. This research will improve our understanding of the epidemiology and evolution of two important but under-addressed bacterial pathogens. The study will determine which subgroups of enteroaggregative Escherichia coli and Salmonella are more likely to cause disease and whether there are subtypes that are associated with life-threatening disease. This is important for the study location, Nigeria, because very little is known about locally-prevalent subtypes within the country or in neighbouring countries. Identification and characterization of predominant subgroups serve as the basis for devising diagnostics to better their detection and surveillance. The findings from this study will also inform vaccine development and vaccine use policy because the most harmful subtypes can be targetted. This research will also determine whether healthy individuals carry these organisms, and if so, to what extent. Understanding healthy carriage is key to determining how these organisms are maintained and transmitted in communities. The study will use molecular methods to characterize the strains in a laboratory to be set up at the University of Ibadan and lead by the African Research Leader Candidate who is co-investigator on this grant. The African Research Leader will additionally build on collaborative links with other regional laboratories and extend some of the expertise built at Ibadan to those labs. The research will therefore build capacity in the area of molecular bacteriology and provide a collaborative link between West African scientists and the Wellcome Trust Sanger Institute, where the principal investigator is located.

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