Genomic insights into sex determination evolution in yam, an important staple food crop

Reductions in the cost of next generation sequencing and expertise required for whole genome assembly and annotation permits improvement of existing assemblies of industrially important models (Chinese hamster ovary cell line - CHO) and sequencing neglected agronomically important species, such as yam. Applying these new technologies, we have produced an improved reference for the CHO lineage, CHO-K1, and generated draft assemblies and annotations for three yam species. Yam is an important staple crop of great cultural and socioeconomic significance to Africa, the Americas, the Caribbean, South Pacific and Asia. I explored the evolutionary history of sex determination in dioecious Dioscorea species, a rare trait found in only 5-6% of angiosperms. We identified the most socio-economically important species, guinea yam (D. rotundata) to be female heterogametic (ZW), and confirmed the related basal species, oni-dokoro (D. tokoro), to be male heterogametic (XY). It is exciting to observe both ZW and XY sex determination systems in Dioscorea, as this indicates turnover of sex determination systems. There has been little study to date comparing plant species in the same genus with different sex determination systems, making Dioscorea a unique opportunity to investigate the turnover of sex determination. Through comparison of these two species, and generation of a draft reference for D. alata, I have begun to elucidate the ancestral state of sex within the genus. Generation of these genomic resources in yam and study of the evolution of sex determination, will assist with breeding programmes that will improve this important staple food crop. Finally, these findings will assist with future studies that aim to improve our fundamental understanding of the mechanisms of recombination and speciation in plants

Transcriptome analysis of the hypothalamic-pituitary-adrenal axis in the experimentally domesticated fox

Variation in activity of the hormonal stress response, or hypothalamic-pituitary-adrenal (HPA) axis, has been associated with different personality traits and coping styles in humans and animals, while its dysregulation has been implicated in psychological disorders. The molecular basis of HPA axis regulation, however, is not yet well understood. Here, foxes selectively bred for tameness or aggression are used as a model to investigate differences in regulation of the HPA axis. Activity of this axis is markedly reduced in tame compared to aggressive foxes, with reduced levels of HPA axis hormones such as adrenocorticotrophic hormone (ACTH) and cortisol both basally and in response to a stressor. Gene expression differences were analyzed using RNA sequencing in the anterior pituitary and adrenal glands of foxes from the tame and aggressive lines, and variant analysis was performed on RNA reads from hypothalamus, anterior pituitary, and adrenal tissues from the same foxes. Pituitary analysis revealed expression differences in genes related to exocytosis and cellular signaling; adrenals analysis identified differences in similar pathways, in addition to genes related to fatty acid and cholesterol synthesis. Variant analysis also implicated cell signaling and exocytosis, as well as ion transport and DNA damage repair. These findings suggest the importance of regulation of hormone release in the control of ACTH and cortisol levels. They also suggest that metabolism of precursors to cortisol, such as fatty acids and cholesterol, may be of greater importance in HPA axis regulation than synthesis of cortisol itself. Finally, in conjunction with previous genomic findings, they suggest an association between DNA repair mechanisms and selection for tameness. These findings provide possible new lines of investigation into biological underpinnings of the phenotypic differences between the tame and aggressive lines of foxes. More broadly, as the tame foxes are considered experimentally domesticated, the findings from this project may prove applicable to HPA axis regulation differences associated with domestication in other species. Additionally, a deeper understanding of HPA axis regulation and dysregulation may be applicable both to variation in the normal population, particularly as related to behavioral traits such as coping styles, and to a number of psychiatric disorders in humans, as well as to behavioral disorders in other species, such as dogs

Learning by Fusing Heterogeneous Data

It has become increasingly common in science and technology to gather data about systems at different levels of granularity or from different perspectives. This often gives rise to data that are represented in totally different input spaces. A basic premise behind the study of learning from heterogeneous data is that in many such cases, there exists some correspondence among certain input dimensions of different input spaces. In our work we found that a key bottleneck that prevents us from better understanding and truly fusing heterogeneous data at large scales is identifying the kind of knowledge that can be transferred between related data views, entities and tasks. We develop interesting and accurate data fusion methods for predictive modeling, which reduce or entirely eliminate some of the basic feature engineering steps that were needed in the past when inferring prediction models from disparate data. In addition, our work has a wide range of applications of which we focus on those from molecular and systems biology: it can help us predict gene functions, forecast pharmacological actions of small chemicals, prioritize genes for further studies, mine disease associations, detect drug toxicity and regress cancer patient survival data. Another important aspect of our research is the study of latent factor models. We aim to design latent models with factorized parameters that simultaneously tackle multiple types of data heterogeneity, where data diversity spans across heterogeneous input spaces, multiple types of features, and a variety of related prediction tasks. Our algorithms are capable of retaining the relational structure of a data system during model inference, which turns out to be vital for good performance of data fusion in certain applications. Our recent work included the study of network inference from many potentially nonidentical data distributions and its application to cancer genomic data. We also model the epistasis, an important concept from genetics, and propose algorithms to efficiently find the ordering of genes in cellular pathways. A central topic of our Thesis is also the analysis of large data compendia as predictions about certain phenomena, such as associations between diseases and involvement of genes in a certain phenotype, are only possible when dealing with lots of data. Among others, we analyze 30 heterogeneous data sets to assess drug toxicity and over 40 human gene association data collections, the largest number of data sets considered by a collective latent factor model up to date. We also make interesting observations about deciding which data should be considered for fusion and develop a generic approach that can estimate the sensitivities between different data sets

Application of omic approaches on the mechanisms of pollutants using Daphnia magna as model species

[eng] Environmental toxicology is undergoing a paradigm shift due to the new concerning environmental reality. Nowadays, to manage subtler and chronic effects of chemicals, either single or mixtures, is an imperative need, especially at low and environmentally relevant concentrations. Not least important is to deal with emerging contaminants (ECs), whose harmful effects in ecosystems and toxicity mechanisms are still unknown. Therefore, new strategies for assessing the toxicity of pollutants with greater environmental relevance must be developed, what requires the application of integrative approaches combining tools from different disciplines. Omic technologies allow the holistic measurement of effects at low levels of biological organization in high throughput platforms, and provide mechanistic data which may become essential in the development and application of more efficient and effective testing strategies. Overall, this thesis aimed to prove the importance of integrating omic and conventional toxicological approaches in order to obtain significant information that helps to unravel any new toxicity mechanism triggered by ECs on the aquatic environment using Daphnia magna as model species. The ECs studied included suspected lipid disruptors (endocrine disrupting compounds, EDCs) and ECs that are known to affect the central nervous system (i.e. neuroactive pharmaceuticals and other chemicals). Different integrative approaches have been developed to assess the toxicity of these compounds by linking effects on reproduction and behavior (individual organism responses), with gene expression changes and its subsequent metabolomic (and thus lipidomic) disruption in D. magna. The ability of EDCs and neuroactive pharmaceuticals to affect reproduction and disrupt lipid homeostasis, as well as the molecular signaling pathways that modulate this disruption, has been addressed throughout the thesis. Within the chapter 2, microarray transcriptomic analysis of D. magna adult females exposed to some EDCs during reproduction was performed, together with the effects on their lipidome by a lipidomic analysis using UHPLC-TOF MS. Common transcriptional mechanisms were identified as energy-related categories, molting and reproduction, and different lipid functional categories. The obtained results allowed to link reproductive effects with changes in lipid profiles and disrupted transference of lipids to eggs in D. magna females. Lipidomic effects of neuroactive pharmaceuticals at environmental concentrations and the driven molecular mechanisms behind them were studied in chapter 3. The hypothesis that serotonin may be involved in regulating lipid dynamics and fecundity responses in D. magna was confirmed by the analysis of the lipidome of genetically tryptophan hydrolase gene knockout clones. Finally, in chapter 4 a targeted metabolomic approached was developed to analyze neurotransmitters in D. magna samples and employed in the study of the effects of neuroactive pharmaceuticals that affected Daphnias’ cognitive behavior. Metabolomic results were linked to the associated transcriptional disruption studied through RNAseq, probing the suitability of these organisms for environmental neurotoxicity studies. Overall, the results obtained throughout this thesis allowed to link transcriptomic signaling pathways with metabolomic effects (lipidomic and neurotransmitter profiles) and with apical responses (reproduction and behavior).[spa] La toxicología ambiental está experimentando un cambio de paradigma debido a la preocupante nueva realidad medioambiental. Hoy en día, gestionar los efectos más sutiles y crónicos de los compuestos químicos, ya sean individualizados o en mezclas, es una necesidad imperiosa, especialmente en concentraciones bajas y relevantes para el medio ambiente. No menos importante es ocuparse de los contaminantes emergentes (EC), cuyos efectos nocivos en los ecosistemas y sus mecanismos de toxicidad aún se desconocen. Por lo tanto, deben elaborarse nuevas estrategias con mayor relevancia ambiental para evaluar la toxicidad de los contaminantes, lo que requiere la aplicación de enfoques integradores que combinen herramientas de distintas disciplinas. Las tecnologías ómicas permiten medidas holística de efectos producidos a bajos niveles de organización biológica en plataformas de alto rendimiento, y proporcionan datos mecanicistas que pueden resultar esenciales para el desarrollo y la aplicación de estrategias de ensayo más eficientes y eficaces. En general, el objetivo de esta tesis ha sido demostrar la importancia de integrar enfoques toxicológicos ómicos con ensayos toxicológicos convencionales a fin de obtener información significativa que ayude a desentrañar cualquier nuevo mecanismo de toxicidad desencadenado por ECs en el medio acuático utilizando Daphnia magna como especie modelo. Los contaminantes estudiados en esta tesis incluyeron aquellos sospechosos de ser disruptores de lípidos (compuestos disruptores endocrinos, EDC) y ECs que se sabe que afectan al sistema nervioso central (es decir, fármacos neuroactivos y otros productos químicos). Se han desarrollado diferentes enfoques integradores para evaluar la toxicidad de estos compuestos vinculando los efectos sobre la reproducción y el comportamiento (respuestas individuales del organismo), con los cambios en la expresión de los genes y su posterior alteración metabolómica (y por tanto lipidómica) en D. magna. A lo largo de esta tesis se ha abordado la capacidad de los EDCs y de los fármacos neuroactivos de afectar a la reproducción y perturbar la homeostasis lipídica, así como a las vías de señalización molecular que modulan esta perturbación. En el capítulo 2, se realizó un análisis transcriptómico mediante microarrays de hembras adultas de D. magna expuestas a algunos EDCs durante su etapa reporductora, y se estudiaron los efectos producidos en su lipidoma mediante un análisis lipidómico utilizando UHPLC-TOF MS. Se identificaron mecanismos transcripcionales comunes descritos con categorías funcionales relacionadas con la energía, la muda y la reproducción, así como diferentes categorías funcionales de lípidos. Los resultados obtenidos permitieron vincular los efectos reproductivos con cambios en los perfiles de lípidos, así como con una alterada transferencia de lípidos de las hembras de D. magna a sus huevos. En el capítulo 3 se estudiaron los efectos lipidómicos de productos farmacéuticos neuroactivos en concentraciones ambientalmente relevantes y los mecanismos moleculares asociados a ellos. La hipótesis de que la serotonina puede participar en la regulación de la dinámica de los lípidos y las respuestas de la fecundidad en D. magna se confirmó mediante el análisis del lipidoma de clones con el gen triptófano hidrolasa silenciado. Por último, en el capítulo 4 se desarrolló un enfoque metabolómico dirigido para analizar neurotransmisores en D. magna y se empleó en el estudio de los efectos de fármacos neuroactivos que afectaban a su comportamiento cognitivo. Los resultados metabólicos se vincularon a la alteración transcripcional asociada estudiada a través del RNAseq, probando la idoneidad de estos organismos para estudios de neurotoxicidad ambiental. En general, los resultados obtenidos a lo largo de esta tesis permitieron vincular las vías de señalización transcriptómica con efectos metabolómicos (perfiles lipidómicos y de neurotransmisores) y con respuestas apicales (reproducción y comportamiento)

Improving host resistance to Fusarium head blight in wheat (Triticum aestivum L.) and Gibberella ear rot in maize (Zea mays L.)

Fusarium head blight (FHB) in wheat and Fusarium (FER) and Gibberella ear rot (GER) in maize are major cereal diseases which reduce yield and contaminate kernels with several mycotoxins. In Europe, these diseases contribute to significant yield gaps and high mycotoxin risks across countries. However, existing management strategies related to agronomic practices are not fully effective, with some of them being cost-prohibitive. Enhancing host plant resistance is additionally required for managing the diseases more effectively and sustainably. Unfortunately, breeding for FHB resistance is challenged by complex interactions with morphological traits and the quantitative nature of the trait. In maize, available genetic resources have not been fully exploited to improve GER resistance in elite materials. In this work, we elucidated the complex interactions between FHB resistance and morphological traits, like plant height (PH) and anther retention (AR) in wheat. The effect of reduced height (Rht) gene Rht24 on AR and the contribution of genomic background (GB) to FHB resistance in semi-dwarf genotypes were also assessed. GB refers to all genomic loci, except major Rht genes, that affect the traits. To achieve this, 401 winter wheat cultivars were evaluated across five environments (location × year combination). All cultivars were genotyped using Illumina 25 K Infinium single-nucleotide polymorphism array. We performed correlation and path coefficient analysis, and combined single and multi-trait genome-wide association studies (GWAS). Our findings revealed significant genotypic correlations and path effects between FHB severity with PH and AR, which were controlled by several pleiotropic loci. FHB severity and PH shared both negatively and positively acting pleiotropic loci, while only positively acting pleiotropic loci were detected between FHB severity and AR. Rht-D1 is a major pleiotropic gene which exerted a negative effect on FHB resistance. These pleiotropic loci contribute to our understanding of the complex genetic basis of FHB resistance, and their exploitation can help to simultaneously select for FHB resistance with PH and AR. Contrary to Rht-D1b, Rht24b had no negative effect on FHB resistance and AR. This exhibits Rht24 as an important FHB-neutral Rht gene which can be integrated into breeding programs. Genomic estimated breeding values (GEBV) were calculated for each cultivar to assess GB. We observed highly negative GEBV for FHB severity within resistant wheat cultivars. Susceptible cultivars exhibited positive GEBV. Genomic prediction has a great potential and can be exploited by selecting for semi-dwarf winter wheat genotypes with higher FHB resistance due to their genomic background resistance. To tackle maize ear rot diseases, refined and stable quantitative trait loci (QTL) harboring candidate genes conferring resistances to FER and GER were identified. The effectiveness of introgression of two European flint landraces, namely Kemater Gelb Landmais (KE) and Petkuser Ferdinand Rot (PE) was evaluated. The prediction accuracy of using line performance as a predictor of hybrid performance for GER resistance was also evaluated within the two landraces. We applied a meta-QTL (MQTL) analysis based on 15 diverse SNP-based QTL mapping studies and performed gene expression analysis using published RNA-seq data on GER resistance. In total, 40 MQTL were identified, of which 14 most refined MQTL harbored promising candidate genes for use in breeding programs for improving FER and GER resistances. 28 MQTL were common to both FER and GER, with most of them being shared between silk (channel) and kernel resistances. This highlights the co-inheritance of FER and GER resistances as well as types of active resistance. Resistance genes can be transferred into elite cultivars by integrating refined MQTL into genomics-assisted breeding strategies. Afterwards, four GER resistant doubled haploid (DH) lines from both KE and PE landraces were crossed with two susceptible elite lines to generate six bi-parental populations with a total of 534 DH lines which were evaluated for GER resistance. GER severity within the six landrace-derived populations were reduced by 39−61% compared to the susceptible elite lines. Moderate to high genetic advance was observed within each population, and the use of KE landrace as a donor was generally more effective than PE landrace. This shows promise in enhancing resistance to GER in elite materials using the European flint landraces as donors. Furthermore, per se performance of 76 DH lines from both landraces was used to predict GER resistance of their corresponding testcrosses (TC). Moderate phenotypic and genomic prediction accuracy between TC and line per se performance was found for GER resistance. This implies that pre-selecting lines for GER resistance is feasible; however, TC should be additionally tested on a later selection stage to aim for GER-resistant hybrid cultivars.Ährenfusariosen (FHB) bei Weizen und Fusarium- (FER) und Gibberella-Kolbenfäule (GER) bei Mais sind wichtige Getreidekrankheiten. In Europa tragen diese Krankheiten zu erheblichen Ertragseinbußen und hohen Mykotoxinrisiken in den einzelnen Ländern bei. Die bestehenden Kontrollstrategien im Zusammenhang mit pflanzenbaulichen Praktiken sind jedoch nicht voll wirksam, und einige von ihnen sind zu kostspielig. Um die Krankheiten wirksamer und nachhaltiger zu bekämpfen, muss deshalb die Resistenz der Wirtspflanzen gestärkt werden. Leider wird die Züchtung auf FHB-Resistenz durch komplexe Wechselwirkungen mit morphologischen Merkmalen und der quantitativen Natur des Merkmals erschwert. Bei Mais wurden die verfügbaren genetischen Ressourcen bisher nicht vollständig genutzt, um die GER-Resistenz von Elitematerial zu verbessern. In dieser Arbeit haben wir die komplexen Wechselwirkungen zwischen FHB-Resistenz und morphologischen Merkmalen wie Wuchshöhe (PH) und Antherenretention (AR) bei Weizen aufgeklärt. Außerdem wurden die Auswirkungen des Gens Rht24 für reduzierte Wuchshöhe (reduced height, Rht) auf AR und der Beitrag des genomischen Hintergrunds (GB) zur FHB-Resistenz bei kurzstrohigen Genotypen untersucht. GB bezieht sich auf alle Genloci, mit Ausnahme der bekannten Rht-Gene, die die jeweiligen Merkmale beeinflussen. Zu diesem Zweck wurden 401 Winterweizensorten in fünf Umwelten (Ort × Jahr-Kombinationen) bewertet. Alle Sorten wurden mit dem Illumina 25k Infinium Single-Nukleotid-Polymorphismus-Array genotypisiert. Wir führten Korrelations- und Pfadkoeffizientenanalysen durch und kombinierten genomweite Assoziationsstudien (GWAS) mit einzelnen bzw. mehreren Merkmalen. Unsere Ergebnisse zeigten signifikante genotypische Korrelationen und Pfadeffekte zwischen FHB-Befallsstärke und PH und AR, die von mehreren pleiotropen Loci kontrolliert wurden. FHB-Befallsstärke und PH hatten sowohl negativ als auch positiv wirkende pleiotrope Loci gemeinsam, während zwischen FHB-Befallsstärke und AR nur positiv wirkende pleiotrope Loci gefunden wurden. Rht-D1 ist ein wichtiges pleiotropes Gen, dessen kurzstrohiges Allel einen negativen Einfluss auf die FHB-Resistenz ausübt. Diese pleiotropen Loci tragen zu unserem Verständnis der komplexen genetischen Grundlage der FHB-Resistenz bei, und ihre Nutzung kann dazu beitragen, gleichzeitig mit verringerter PH und AR auf FHB-Resistenz zu selektieren. Im Gegensatz zu Rht-D1b hatte Rht24b keine Auswirkungen auf FHB-Resistenz und AR. Dies zeigt, dass Rht24 ein wichtiges FHB-neutrales Rht-Gen ist, das in Zuchtprogramme integriert werden kann. Zur Bewertung des GB wurden für jede Sorte genomisch geschätzte Zuchtwerte (GEBV) berechnet. Bei resistenten Weizensorten beobachteten wir einen stark negativen GEBV für FHB-Befallsstärke. Anfällige Sorten wiesen einen positiven GEBV auf. Die genomische Vorhersage hat ein großes Potenzial und kann durch die Selektion von kurzstrohigen Winterweizengenotypen mit höherer FHB-Resistenz aufgrund ihrer genomischen Hintergrundresistenz genutzt werden. Zur Bekämpfung von Mais-Kolbenfäule wurden stabile quantitative Merkmalsloci (QTL) identifiziert, die Kandidatengene für Resistenzen gegen FER und GER beherbergen. Die Wirksamkeit der Introgression von Resistenzen aus zwei europäischen Flint-Landrassen, nämlich "Kemater Gelber Landmais" (KE) und "Petkuser Ferdinand Rot" (PE), wurde bewertet. Die Vorhersagegenauigkeit der Verwendung der Linienleistung als Vorhersage für die Hybridleistung bei der GER-Resistenz wurde ebenfalls innerhalb der beiden Landrassen bewertet. Wir haben eine Meta-QTL-Analyse (MQTL) auf der Grundlage von 15 SNP-basierten QTL-Kartierungsstudien durchgeführt und eine Genexpressionsanalyse anhand veröffentlichter RNAseq-Daten zur GER-Resistenz vorgenommen. Insgesamt wurden 40 MQTL identifiziert, von denen die 14 stabilsten MQTL vielversprechende Kandidatengene für den Einsatz in Zuchtprogrammen zur Verbesserung von FER- und GER-Resistenzen enthielten. 28 MQTL waren sowohl für FER- als auch GER-Resistenz verantwortlich, wobei die meisten sowohl für Narbenfaden- als auch Körnerresistenz verantwortlich waren. Die Resistenz kann in Elitesorten übertragen werden, indem präzisierte MQTL in genomgestützte Züchtungsstrategien integriert werden. Anschließend wurden vier GER-resistente doppelhaploide (DH) Linien aus KE- und PE-Landrassen mit zwei anfälligen Elitelinien gekreuzt, um sechs bi-parentale Populationen mit insgesamt 534 DH-Linien zu erzeugen, die mehrortig auf GER-Resistenz untersucht wurden. Der GER-Befallsstärke war bei den sechs von Landrassen abgeleiteten Populationen im Vergleich zu den anfälligen Elitelinien im Mittel um 39-61% reduziert. Durch Einkreuzung der jeweiligen Population kann ein mäßiger bis hoher genetischer Fortschritt erzielt werden, die Verwendung der KE-Landrasse als Spender war effektiver als die der PE-Landrasse. Dies ist ein vielversprechender Ansatz, um die Resistenz von Elitematerialien gegen GER zu verbessern. Darüber hinaus wurde die Leistung von 76 DH-Linien aus beiden Landrassen zur Vorhersage der GER-Resistenz der entsprechenden Testkreuzungen (TC) verwendet. Für die GER-Resistenz wurde eine mäßige phänotypische und genomische Vorhersagegenauigkeit zwischen TC und der Leistung der Linie festgestellt. Eine Vorselektion von Linien auf GER-Resistenz ist deshalb möglich; die Leistung der TC sollte jedoch in einer späteren Selektionsphase zusätzlich getestet werden, um GER-resistente Hybridsorten zu erhalten

Genomic characterisation of Alzheimer’s disease risk genes using long-read sequencing

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterised by progressive intracellular accumulation of hyperphosphorylated tau and extracellular deposition of beta-amyloid. It affects over 50 million people worldwide with numbers expecting to triple by 2050. Despite recent success in identifying genetic risk factors for AD, the mechanisms underpinning disease progression remain unknown. There is increasing evidence for altered transcriptional regulation and RNA splicing in the development of AD pathology. However, current studies exploring isoform diversity in the AD brain are constrained by the inherent limitations of standard short-read RNA-sequencing approaches, which fail to capture full-length transcripts critical for transcriptome assembly. The primary aim of this thesis was to utilise two long-read sequencing approaches, Pacific Biosciences isoform sequencing and Oxford Nanopore Technologies nanopore cDNA sequencing, to examine isoform diversity and transcript usage in the cortex, and identify alternative splicing events associated with AD pathology in a transgenic model of tau pathology (rTg4510). By generating long reads that span full-length transcripts, our studies revealed widespread RNA isoform diversity with unprecedented detection of novel transcripts not present in existing genome annotations. We further performed ultra-deep targeted long-read sequencing of 20 AD-risk genes, identifying robust expression changes at the transcript level associated with tau accumulation in the cortex. Our analyses provide a systematic evaluation of transcript usage, even in the absence of gene-level expression alterations, and highlight the importance of alternative RNA splicing as a mechanism underpinning gene regulation in the development of tau pathology. Finally, this thesis presents a laboratory and bioinformatics pipeline for the systematic characterisation of isoform diversity and alternative splicing using long-read sequencing. The data generated as part of this research have implications for our understanding of the mechanisms driving the development of tau pathology, and represent a valuable resource to the wider research community

Insights into transcriptional regulation from natural and induced variation in closely related species

Despite years of research, numerous aspects of gene regulatory mechanisms and their contributions to shaping phenotypic characteristics remain elusive. In this thesis, I gain insights into mechanisms of transcriptional regulation, focusing particularly on the binding activity of transcription factors (TFs). In the comprised projects and analyses, I use models of closely related mouse species, as in vivo systems that leverage a pool of molecular variation, in order to study the activity and roles of TF binding within wider functional contexts. The molecular variation is manifested as variation in the sequence context and/or in the binding activity of the TFs, and it may be naturally fixed by evolution among the different species, and/or induced variation among different conditions, for example as a result to carcinogen exposures. The reflection of the underlying molecular variation in the functional roles of TF binding is also tied to some extent to functional outputs and is examined as part of general functional contexts, such as higher-order genome organisation or the neoplastic transformation of a tumour cell in cancer development. In the first part of the thesis, I explore the evolutionary dynamics of the CTCF (CCCTC-binding factor) binding among mouse species and its interplay with the establishment and maintenance of topologically associating domains (TADs). TADs make up a level of the 3D genome organisation that dictates formation of regulatory landscapes. Although it is known that CTCF plays an important role in TAD formation, evidence of TAD boundaries being robust to CTCF depletion interferences has confounded the understanding of its exact role. My study reveals the occurrence of dynamically evolving clusters of neighbouring CTCF sites at the boundaries of TADs, which contribute to the resilience and flexibility of the TAD structures. My findings also highlight the necessity of examining the binding of CTCF not only as individual binding sites, but also as an ensemble of potentially functional sites that can exert their actions synergistically, additively or interchangeably, and contribute to fostering phenotypic features. In the second part of the thesis, I present a project I worked on as a member of a collaborative effort of the Liver Cancer Evolution (LCE) consortium. The project makes use of a model of chemically induced liver carcinogenesis in different mouse species, which offer a controlled biological system with natural genomic and epigenomic variation. Here, I characterise the expression profile of the cell-of-origin, the hepatocyte, of chemically induced liver tumours and the shift of its cell-type specific phenotype along neoplastic transformation, which is underlined by gene dysregulation. In addition, I further explore associations of gene dysregulation in the cell-of-origin with mutagenesis (induced genetic variation as a result of carcinogen exposure) in liver TF binding sites that potentially underlie the onset of hepatocyte dedifferentiation in tumour development. My results disentangle patterns of mutation accumulation among distinct categories of TF binding sites based on their functional characteristics, such as combinatorial binding or association with with cis-regulatory elements. Also, they reveal sets of hyper-mutated TF binding sites in liver tumours, which are functionally enriched for liver-specific biological processes, cellular stress response and abnormal liver phenotype, and they are associated with dysregulated genes with hepatocyte-specific functions. Finally, these findings also highlight the necessity of studying the activity of TFs bound both in a singleton manner and in concert and within their broader functional context.EMBL International PhD Programm

Transcriptomic analysis of sheep macrophages and their response to Lipopolysaccharide

Sheep are ruminant animals, highly adapted to exploit pastures of low biological value and an economically important livestock species. They also provide a useful animal model for multiple areas of human medicine. Their productivity is compromised by many viral, bacterial, fungal, protozoan and helminthic pathogens. The innate immune system evolves under stringent selective pressure from pathogens. In this project, I aimed to identify genes involved in innate immunity in sheep by dissecting the transcriptome of sheep macrophages. The analysis focussed on identification of genes that may be associated with either disease susceptibility or resistance traits. The project formed part of the broader transcriptomic atlas for sheep. A method was established and validated for the production of sheep bone marrow derived macrophages (BMDMs) grown in macrophage colony‐stimulating factor (CSF1). These cells responded to bacterial Lipopolysaccharide (LPS), an archetypal agonist of the pattern recognition receptor, TLR4, with induction of inflammatory cytokines, but unlike rodent macrophages, sheep produced no nitric oxide. Bone marrow‐derived macrophages were produced from male (n=3) and female (n=3) Scottish Blackface X Texel animals used in The Sheep Atlas project, and mRNA was isolated from the cells at 0, 2, 4, 7 and 24 hours following stimulation. Two different protocols of mRNA preparation were used. For 0 and 7 hour samples, samples were depleted of rRNA, and RNA‐Seq was carried out at a depth of 100 million reads. For all samples in the time course (including 0 and 7), mRNA was prepared by polyA selection, and RNA‐Seq was performed at lower depth (25 million reads) and a detailed analysis of the different outcomes is presented. Two pipelines, Kallisto and StringTie were used to identify and quantify transcripts in the LPS time course transcriptomic data, along with other subsets of innate immune cells from the wider atlas. The former pipeline provides quantitative known transcript expression estimates, the latter generates novel transcript and gene models. Analysing the transcriptional signatures of these samples provided insight into the metabolic, regulatory and innate response pathways that sheep share with other animals, with both Myeloid differentiation primary response 88 protein (MYD88) dependent and independent pathways being activated following LPS stimulation, with hundreds of the same downstream cytokines being highly expressed in response, such as TNF and many interleukins as seen in other species innate responses. It has also highlighted aspects of the response that separate sheep from other animals, such as their metabolism and biosynthesis of steroid and tryptophan as well as demonstrating differences in specific expression of certain genes. Two highly regulated and expressed genes noted during the peak response at 7hrs (ENSOARG00000005159 and ENSOARG00000006889), were both assigned a functional annotation as being the protease inhibitor Serpin family B member 2 gene (SERPINB2). This appears to be a gene duplication. This sheep expression profile of SERPINB2 is shared with mice but distinct from humans and pigs. Similar to other animals, individual animals were found to vary markedly in their transcriptional response to LPS, demonstrating hyper, early and delayed responses between the individuals. The current reference transcriptome OarV3.1 contains 28757 transcripts, of which only 18488 are functionally annotated. More than 85% of all reference transcripts were detected in sheep macrophages and over 300 candidate annotations for genes identified only by Ensembl sheep (Ovis aries) gene ID (ENSOARG) numbers were manually assigned a functional annotation by clustering co‐expression estimates using the network analysis tool, Miru and inferring function by ‘guilt by association’ of these unknown genes which demonstrated similar expression profiles to those with known function. The nearest orthologues and synteny with other species were used to validate these suspected annotations. A select few examples include PYD and CARD domain containing gene (PYCARD), all the early growth response factors and many of the C‐X‐C Motif chemokines. In summary, this project identified multiple sheep‐specific aspects of the innate immune response, whilst assigning hundreds of genes a functional annotation and detecting new transcript models for multiple known genes. Many regulated, highly expressed, multi exoned, novel gene models have also been identified which are worthy of future investigation