Advances in quantitative microscopy

Microscopy allows us to peer into the complex deeply shrouded world that the cells of our body grow and thrive in. With the emergence of automated digital microscopes and software for anlysing and processing the large numbers of image that they produce; quantitative microscopy approaches are now allowing us to answer ever larger and more complex biological questions. In this thesis I explore two trends. Firstly, that of using quantitative microscopy for performing unbiased screens, the advances made here include developing strategies to handle imaging data captured from physiological models, and unsupervised analysis screening data to derive unbiased biological insights. Secondly, I develop software for analysing live cell imaging data, that can now be captured at greater rates than ever before and use this to help answer key questions covering the biology of how cells make the decision to arrest or proliferate in response to DNA damage. Together this thesis represents a view of the current state of the art in high-throughput quantitative microscopy and details where the field is heading as machine learning approaches become ever more sophisticated.Open Acces

Genome-wide microscopy screening identifies links across processes including a conserved connection between DNA damage control and the microtubule cytoskeleton

Previous PhD students in the lab created a method for large-scale, high-content microscopy screening of a cell library consisting of over 3000 single mutant strains of the fission yeast, Schizosaccharomyces pombe. Each strain has one nonessential gene knocked-out, allowing investigation of the resulting phenotypes. I report the implementation and completion of this screen; developing methods to ensure reliable and accurate results through inclusion of many controls across multiple screening repeats. In total, over 4.5 million images from approximately 19 000 biologically independent cell populations were imaged and analysed. All strains screened contained GFP-labelled tubulin (GFP-Atb2) allowing visualisation of the microtubule polymer network and its organisation in cells, a feature that is conserved across eukaryotes and simplified in S. pombe, making it easy to study. Examination of cell outlines and microtubule patterns was used to study three cell processes: the shape of cells, the organisational pattern of interphase microtubules and the cell cycle stage of cells, as judged by microtubule pattern. Comparison with extensive data from wild-type cells led to the identification of 262 factors that influence one or more of these cell processes. I go on to biologically validate some of the outcomes from the screen, leading to a publication in Developmental Cell reporting the screen, its findings and the online genomic resource SYSGRO. I then focus on a group of mutants that suggest a connection between the DNA damage response (DDR) and microtubule organisation. From here I show that the DDR induces elongation of microtubule bundles in response to the DDR kinases, ATM and ATR. I begin to reveal factors that may mediate this response and finally, I provide evidence to suggest that the same mechanism is conserved in cultured human cells (Hc3716-hTERT), which may go some way to explaining clinical results showing a beneficial effect of microtubule destabilisation in conjunction with cancer therapies

Deep mutational scanning of mammalian loci using CRISPR-Cas9 and multiplex HDR

Functional consequences of genetic variants are best studied in their endogenous chromosomal context. Gene editing by homology-directed repair can introduce such predetermined genetic changes into chromosomal DNA. In this thesis, I develop methods to generate tens to hundreds of genetic variants, expressed from a native chromosomal context, and simultaneously evaluate their phenotypic impact. This approach involves repair of Cas9-derived double strand breaks (DSBs) from oligonucleotide repair template libraries containing controlled levels of nucleotide heterogeneity. Cell populations are then purified based on a phenotypic assay and subjected to deep amplicon sequencing at the target site to link genotype with phenotype. In the first chapter, I developed a bioinformatics pipeline for the processing of Illumina sequencing reads containing nucleotide variants, and validate this pipeline in silico. As a proof-of-principle, in the second chapter I then introduced nucleotide variants across 8 codons of a chromosomal GFP transgene in mouse embryonic stem cells. The functional impact of these variants was quantified, with the results benchmarked against an existing episomal dataset, and by in silico modelling of mutant protein structure. In the final chapter, I applied this pipeline to analyse a CRISPR deep mutational scanning dataset incorporating all possible amino acid substitutions within a region of β-catenin, a component of the Wnt signalling pathway, that is a mutational hotspot in many types of cancer. The functional impact of these clinically relevant variants was assessed using a fluorescent reporter of Wnt signalling. By combining the resulting functional scores with mutational signature data from genome sequencing of different tumour types, I finally dissect the relative contribution of mutational bias and natural selection to the different patterns of amino acid substitutions found in different tumour types

Deciphering causal genetic determinants of red blood cell traits

Les études d’association pan-génomiques ont révélé plusieurs variants génétiques associés à des traits complexes. Les mesures érythrocytaires ont souvent fait l’objet de ce genre d’études, étant mesurées de façon routinière et précise. Comprendre comment les variations génétiques influencent ces phénotypes est primordial étant donné leur importance comme marqueurs cliniques et leur influence sur la sévérité de plusieurs maladies. En particulier, des niveaux élevés d’hémoglobine fœtal chez les patients atteints d’anémie falciforme est associé à une réduction des complications et une augmentation de l’espérance de vie. Néanmoins, la majorité des variants génétiques identifiés par ces études tombent à l’intérieur de régions génétiques non-codantes, augmentant la difficulté d’identifier des gènes causaux. L’objectif premier de ce projet est l’identification et la caractérisation de gènes influençant les traits complexes, et tout particulièrement les traits sanguins. Pour y arriver, j’ai tout d’abord développé une méthode permettant d’identifier et de tester l’effet de gènes knockouts sur les traits anthropométriques. Malgré un échantillon de grande taille, cette approche n’a révélé aucune association. Ensuite, j’ai caractérisé le méthylome et le transcriptome d’érythroblastes différentiés à partir de cellules souches hématopoïétiques et identifié plusieurs gènes potentiellement impliqués dans les programmes érythroïdes fœtaux et adultes. Par ailleurs, j’ai identifié plusieurs micro-ARNs montrant des motifs d’expression spécifiques entre les stages fœtaux et adultes et qui sont enrichis pour des cibles exprimées de façon opposée. Finalement, j’ai identifié plusieurs variants génétiques associés à l’expression de gènes dans les érythroblastes (eQTL). Cette étude a permis d’identifier des variants associés à l’expression du gène ATP2B4, qui encode le principal transporteur de calcium des érythrocytes. Ces variants, qui sont également associés à des traits sanguins et à la susceptibilité à la malaria, tombent dans un élément d’ADN spécifique aux cellules érythroïdes. La délétion de cet élément par le système CRISPR/Cas9 induit une forte diminution de l’expression du gène et une augmentation des niveaux de calcium intracellulaires. En conclusion, des échantillons de génotypages exhaustifs seront nécessaires pour étudier l’effet de gènes knockouts sur les traits complexes. Les érythroblastes montrent de grandes différences au niveau de leur méthylome et transcriptome entre les différents stages développementaux. Ces différences influencent potentiellement la régulation de l’hémoglobine fœtale et impliquent de nombreux micro-ARNs et régions régulatrices non-codantes. Finalement, l’exemple d’ATP2B4 montre qu’intégrer des études épigénomiques, transcriptomiques et des expériences d’édition de génome est une approche puissante pour caractériser des variants génétiques non-codants. Par ailleurs, ces résultats impliquent ATP2B4 dans l’hydratation des érythroblastes, qui est associé à la susceptibilité à la malaria et la sévérité de l’anémie falciforme. Cibler ATP2B4 de façon thérapeutique pourrait avoir un impact majeur sur ces maladies qui affectent des millions d’individus à travers le monde.Genome-wide association studies (GWAS) have revealed several genetic variants associated with complex phenotypes. This is the case for red blood cell (RBC) traits, which are particularly amenable to GWAS as they are routinely and accurately measured. Understanding RBC trait variation is important given their significance as clinical markers and modifiers of disease severity. Notably, increased fetal hemoglobin (HbF) production in sickle cell disease (SCD) patients is associated with a higher life expectancy and decreased morbidity. Nonetheless, most variants identified through GWAS fall in non-coding regions of the human genome, increasing the difficulty of identifying causal links. The main goal of this project was to identify and characterize genes influencing complex traits, and in particular RBC phenotypes. First, I developed an approach to identify and test potential gene knockouts affecting anthropometric traits in a large sample from the general population, which did not yield significant associations. Then, I characterized the DNA methylome and transcriptome of erythroblasts differentiated ex vivo from hematopoietic progenitor stem cells (HPSC), and identified several genes potentially implicated in fetal and adult-stage erythroid programs. I also identified microRNAs (miRNA) that show specific developmental expression patterns and that are enriched in inversely expressed targets. Finally, I mapped expression quantitative trait loci (eQTL) in erythroblasts, and identify erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of RBCs. These genetic variants are associated with RBC traits and malaria susceptibly, and overlap an erythroid-specific enhancer of ATP2B4. Deletion of this regulatory element using CRISPR/Cas9 experiments in human erythroid cells minimized ATP2B4 expression and increased intracellular calcium levels. In conclusion, large and comprehensive genotyping datasets will be necessary to test the role of rare gene knockouts on complex phenotypes. The transcriptomes and DNA methylomes of erythroblasts show substantial differences correlating with their developmental stages and that may be implicated in HbF production. These results also suggest a strong implication of erythroid enhancers and miRNAs in developmental stage specificity. Finally, characterizing the erythroid-specific enhancer of ATP2B4 suggest that integrating epigenomic, transcriptomic and gene editing experiments can be a powerful approach to characterize non-coding genetic variants. These results implicate ATP2B4 in erythroid cell hydration, which is associated with malaria susceptibility and SCD severity, suggesting that therapies targeting this gene could impact diseases affecting millions of individuals worldwide

Identifying therapeutic targets in glioma using integrated network analysis

Gliomas are the most common brain tumours in adult population with rapid progression and poor prognosis. Survival among the patients diagnosed with the most aggressive histopathological subtype of gliomas, the glioblastoma, is a mere 12.6 months given the current standard of care. While glioblastomas mostly occur in people over 60, the lower-grade gliomas afflict themselves upon individuals in their third and fourth decades of life. Collectively, the gliomas are one of the major causes of cancer-related death in individuals under fortyin the UK. Over the past twenty years, little has changed in the standard of glioma treatment and the disease has remained incurable. This study focuses on identifying potential therapeutic targets in gliomasusing systems-level approaches and large-scale data integration.I used publicly available transcriptomic data to identify gene co-expression networks associated with the progression of IDH1-mutant 1p/19q euploid astrocytomas from grade II to grade III and high-lighted hub-genes of these networks, which could be targeted to modulate their biological function. I also studied the changes in co-expression patterns between grade II and grade III gliomas and identified a cluster of genes with differential co-expression in different disease states (module M2). By data integration and adaptation of reverse-engineering methods, I elucidated master regulators of the module M2. I then sought to counteract the regulatory activity by using drug-induced gene expression dataset to find compounds inducing gene expression in the opposite direction of the disease signature. I proposed resveratrol as a potentially disease modifying compound, which when administered to patients with a low-grade disease could potentially delay glioma progression.Finally, I appliedanensemble-learning algorithm on a large-scale loss-of-function viability screen in cancer cell-lines with different genetic backgrounds to identify gene dependencies associated with chromosomal copy-number losses common intheglioblastomas. I propose five novel target predictions to be validated in future experiments.Open acces

Genetics and Genomics of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is a severe and progressive disorder affecting the blood vessels in the lungs. Typically, symptoms first appear at around 30–40 years of age and, without treatment, can lead to fatal heart disease within a few years. Genetic studies over the past decade have identified numerous genes that contribute to disease progression but, for many sufferers, the underlying genetic cause remains elusive. The collection of reviews and original research articles contained within this book provide an overview of recent advancements in understanding the genetic risk factors for pulmonary arterial hypertension. We further examine the emerging interplay between genetic variants and clinical outcomes, providing a framework for new treatments and improved patient care