High-throughput sequencing has greatly influenced the amount of data produced and biological questions asked and answered. Sequencing approaches have also enabled rapid development of related technological fields such as single-cell and spatially resolved expression profiling. The introductory parts of this thesis give an overview of the basic molecular and technological apparatus needed to analyse the transcriptome in cells and tissues. This is succeeded by a summary of present investigations that report recent advancements in RNA profiling. RNA integrity needs to be preserved for accurate gene expression analysis. A method providing a low-cost alternative for RNA preservation was reported. Namely, a low concentration of buffered formaldehyde was used for fixation of human cell lines and peripheral blood cells (Paper I). The results from bulk RNA sequencing confirmed gene expression was not negatively impacted with the preservation procedure (r2&gt;0.88) and that long-term storage of such samples was possible (r2=0.95). However, it is important to note that a small population of cells overexpressing a limited amount of genes can skew bulk gene expression analyses making them sufficient only in carefully designed studies. Therefore, gene expression should be investigated at the single cell resolution when possible. A method for high-throughput single cell expression profiling termed microarrayed single-cell sequencing was developed (Paper II). The method incorporated fluorescence-activated cell sorting, sample deposition and profiling of thousands of barcoded single cells in one reaction. After sample attachment to a barcoded array, a high-resolution image was taken which linked the position of each array barcode sequence to each individual deposited cell. The cDNA synthesis efficiency was estimated at 17.3% while detecting 27,427 transcripts per cell on average. Additionally, spatially resolved analysis is important in cell differentiation, organ development and pathological changes. Current methods are limited in terms of throughput, cost and time. For that reason, the spatial transcriptomics method was developed (Paper III). Here, the barcoded microarray was used to obtain spatially resolved expression profiles from tissue sections using the same imaging principle. The mouse olfactory bulb was profiled on a whole-transcriptome scale and the results showed that the expression correlated well (r2=0.94-0.97) as compared to bulk RNA sequencing. The method was 6.9% efficient, reported signal diffusion at ~2 μm and accurately deconvoluted layer-specific transcripts in an unbiased manner. Lastly, the spatial transcriptomics concept was applied to profile human breast tumours in three dimensions (Paper IV). Unbiased clustering revealed previously un-annotated regions and classified them as parts of the immune system, providing a detailed view into complex interactions and crosstalk in the whole tissue volume. Spatial tumour classification divulged that certain parts of the tumour clearly classified as other subtypes as compared to bulk analysis providing useful data for current practice diagnostics. The last part of the thesis discusses a look towards the future, how the presented methods could be used, improved upon or combined in translational research. / <p>QC 20170109</p>

<p>Lymphomas are a heterogeneous group of hematological malignancies composed of diseases with diverse molecular origins and clinical outcomes. Derived from immune cells of lymphoid origin, lymphoma can arise from lymphoid cells present anywhere in the body, from the spleen and lymph nodes to peripheral sites like the liver and intestines. Current strategies for lymphoma diagnosis involve primarily histopathological examinations of the tumor biopsy, including cytogenetics and immunophenotyping. As more data becomes available, diagnoses may increasingly depend on genomic features that define each disease. Classification of lymphoid neoplasms is generally based on the cell of origin, or the lineage of the normal cell that the cancer is thought to arise from. Lymphomas can be classified into dozens of distinct diagnostic entities, though any two patients with the same diagnosis may have very different outcomes and molecular underpinnings, so we need to understand both the commonalities of patients with the same disease and the unique features that may require personalized treatment strategies. Patient prognosis in lymphoma depends greatly on the type of lymphoma, ranging from nearly curable diseases with over 90% five-year survival rates, to most patients dying in the first year in the worse entities. Greater clarity is needed in the role of the underlying genomics that contribute to these variable treatment responses and clinical outcomes. </p><p>Next-generation sequencing approaches allow us to delve into the molecular underpinnings of lymphomas, in order to gain insight about the origin and evolution of these diseases. High-throughput sequencing protocols allow us to examine the whole genome, exome, epigenome, or transcriptome of cancer cells in tens to hundreds of patients for each disease. As cost of sequencing is reduced, and the ability to generate more data increases, we face increasing computational challenges to both process and interpret the wealth of data available in cancer genomics. Developing efficient and effective bioinformatics tools is necessary to transform billions of sequencing reads into actionable hypotheses on the role of certain genes or biological pathways in a specific cancer type or patient. </p><p>In this dissertation, I present several strategies and applications of integrative computational genomics in lymphoma, with contributions throughout the research process, from development of initial assays and quality control strategies for the sequencing data, to joint analysis of clinical and genomic data, and finally through follow-up experimental models for lymphoma. </p><p>First, I focus on two rare T cell lymphomas, hepatosplenic T cell lymphoma (HSTL) and enteropathy associated T cell lymphoma (EATL), which are both diseases with very poor clinical outcomes and a previous dearth of knowledge on the genetic basis of the diseases. We define the somatic mutation landscape of HSTL, through application of exome sequencing and find SETD2 to be the most highly mutated gene. We further utilize the exome sequencing data to investigate copy number alterations and show a significant survival difference between cases with and without certain arm-level copy number alterations. Knockdown of SETD2 in an HSTL cell line, followed by RNA sequencing, demonstrates the role of SETD2 loss in proliferation and cell cycle changes, linking the SETD2 mutations to a potential oncogenic mechanism. Furthermore, we investigate the potentially targetable mutations in the JAK-STAT pathway and demonstrate oncogenic downstream molecular phenotypes and potential druggability of these mutations. In the enteropathy associated T cell lymphoma study, we apply exome and RNA sequencing to a large EATL cohort. Our findings show a significant role for loss of function mutations in chromatin modifiers and JAK-STAT signaling genes. EATL can be separated into two subtypes, Type I and Type II, which we show to have convergent genomic features, in the face of divergent gene expression. RNA sequencing data defines a distinct separation between the two subtypes. Delving further into the role of SETD2 in these T cell lymphomas, we generate a mouse model with a conditional knockout of SETD2 in T cells and demonstrate a role for SETD2 in altering the lineage development of T cells. </p><p>To understand more about why certain genetic abnormalities are recurrent in some disease entities and not others, we turn to the cell of origin for clues. We pair two different lymphomas, Burkitt lymphoma and mantle cell lymphoma, with their associated cells of origin, germinal center B cells and naive B cells. These closely related cell types have much in common as B cells, but from studies of their transcriptomes, we know that there are many molecular differences that distinguish the two. In this work, after looking more closely at mantle cell lymphoma genomics, we look at the underlying chromatin markers that define the epigenomes of these B cells. We test the association between chromatin markers and mutation rates of genes between these two cell types and lymphomas, and find that genes with more open chromatin may have a higher mutation rate, when comparing closely related cells and lymphomas. Finally, I present my work on developing an RNA sequencing based strategy for defining the complete transcriptome of diffuse large B cell lymphoma (DLBCL). Gene expression profiling with microarray has shown the existence of two subtypes in DLBCL, activated B cell like (ABC) and germinal center B cell like (GCB). However, the role for non-coding RNAs, alternative splicing, and mutations, in these two subtypes and the larger group is previously not well understood. We develop a strand-specific RNA sequencing strategy that will allow the investigation of the total RNA transcriptome in DLBCL, including microRNAs, lncRNAs, and other important non-coding RNAs. Furthermore, we show that RNA sequencing can be used to distinguish the two subtypes, including through RNA sequencing based mutation calls, as well as through differentially expressed lncRNAs that we define for the first time in DLBCL.</p><p>Broadly, this dissertation contributes novel findings in the field of lymphoma genomics, as well as presenting a framework for computational integrative genomics that can guide future studies. The heterogeneity of lymphoma across cases requires us to dive deep into individual diseases, even rare ones, as well as appreciate the similarities and differences across lymphomas. To improve diagnoses, prognoses, and treatment options, we need to understand the molecular origins of lymphoma. Using a range of molecular and computational approaches, we can move closer to true personalized medicine at the genomic level.</p> / Dissertation

Rice blast disease is caused by the filamentous ascomycete fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. The pathogen elaborates a specialized infection structure called the appressorium. The morphological and physiological transitions that lead to appressorium formation of M. oryzae are stimulated through perception of environmental signals and are tightly regulated by cell cycle checkpoints. External stimuli are internalized by a variety of intracellular MAP kinase signaling pathways, and the major pathway regulating appressorium morphogenesis and plant infection is the Pmk1 MAP kinase signaling pathway. The central kinase, Pmk1, is required for appressorium morphogenesis and the homeobox and C2/H2 Zn-finger domain transcription factor, called Mst12, is required for appressorium formation and tissue invasion. The Mst12 null mutant is able to form melanised appressoria, but it is non-pathogenic. To understand the mechanism of appressorium morphogenesis and penetration peg formation, genome-wide comparative transcriptional profiling analysis was performed for the Δpmk1 and Δmst12 mutant using RNA-seq and HiSeq 2000 sequencing. This thesis reports the identification of gene sets regulated by the Pmk1 signalling pathway and defines the sub-set of these genes regulated by Mst12. I show that a hierarchy of transcription factors is likely to operate downstream of Pmk1 to regulate the main processes required for appressorium morphogenesis and plant infection. I also report the role of Mst12 in cytoskeletal re-organisation and show that it is necessary for septin-dependent F-actin polymerisation at the base on the appressorium prior to plant infection. This is consistent with the major transcriptional changes observed by RNA-seq. The thesis also reports experiments that strongly suggest that appressorium mediated plant penetration is regulated by an S-phase checkpoint which operates independently of the conventional DNA damage and repair response, and the Cds1 and Chk1 checkpoint kinases. Transcriptional profiling results are consistent with the S-phase checkpoint operating downstream of the Pmk1 MAP kinase signalling pathway. An integrated model for the operation of the Pmk1/Mst12 signalling pathways and the hierarchical control of appressorium morphogenesis in the rice blast fungus is presented.

La salinité du sol affecte la croissance des plantes glycophytes telle que Arabidopsis thaliana et le riz. Chez les plantes vasculaires, les racines sont composées de divers types de cellules organisées en cercles concentriques. Chaque type de cellules racinaires possède une fonction biologique spécifique et coordonnée avec les autres cellules qui composent cette même racine. Il est probable que la réponse des gènes au stress salin soit spécifique du type cellulaire, ce qui ne peut être révélé par des études à l'échelle de l'organe entier. Afin d'étudier les réponses spécifiques, notre approche a été de générer des profils de transcriptome pour deux types de cellules racinaires chez les plantes modèles, Arabidopsis et riz. Les deux types de cellules étudiées ont été choisis en raison de leur rôle possible soit dans le stockage du sodium dans les cellules corticales, soit dans son transport dans les cellules du péricycle chez Arabidopsis ou du cylindre central chez le riz. Des plantes exprimant la protéine fluorescente verte (GF) spécifiquement dans un type de cellule racinaire furent utilisées pour cette analyse. Les cellules ont donc pu être isolées chez le riz et Arabidopsis grâce à la technique de cytométrie en flux.L'analyse du transcriptome des cellules du péricycle et du cylindre central montrent que les cellules corticales sont plus réactives au stress salin et qu'une large majorité des gènes est sous–exprimée chez les deux plantes modèles. D'après les analyses d'expression des cellules du cortex d'Arabidopsis, trois voies métaboliques sont significativement sous-exprimées en réponse au stress salin: la voie de biosynthèse des phénylpropanoïdes, le transport de l'eau and le métabolisme secondaire. La régulation de gènes impliqués dans le transport de l'eau et des nutriments démontre l'importance des cellules corticales dans le mouvement des solutés. Chez le riz, les profils des deux types cellulaires étudiés révèlent une forte réaction de défense ; en effet le métabolisme protéique et la régulation de la transcription sont fortement sous-exprimés dans les cellules corticales alors que les cellules du cylindre central modifient et activent les gènes correspondant à divers catégories fonctionnelles telles que la réplication de l'ADN et le transport. Des gènes candidats ont été sélectionnés dans les deux types cellulaires d'Arabidopsis. Des lignées mutantes n'exprimant pas ces gènes ont été testées en stress salin dans des conditions hydroponiques. Les résultats ont révélé un phénotype accumulant moins de sodium dans les parties aériennes (20% par rapport au génotype sauvage) pour certaines de ces lignées mutantes. Ce travail est la première étude de transcriptome utilisant des types spécifiques de cellules racinaires chez le riz. L'identification de gènes et voies métaboliques répondant au stress salin dans le cortex et le cylindre central de la racine ouvre de nouveaux axes de recherche et va permettre d'élucider la complexité des processus biologiques d'adaptation au stress salin. / Soil salinity reduces the growth of glycophytic plants such as Arabidopsis thaliana and rice. In vascular plants, roots are organized into concentric layers of cells and each layer has a specific biological function coordinated with other cell types in the root. Therefore, genes differentially expressed in response to a salt stress are also likely to be changing only in specific cell types, and thus may not be revealed at the organ level. In order to identify novel salt-responsive genes, cell-type specific transcriptomic approaches were undertaken in Arabidopsis thaliana and rice, with application of physiologically reasonable salt stress (50mM) over 48 hours. Two cell-types from the root were chosen in both species for their potential role in salt storage and transport: cortical and pericycle/stelar cells respectively. Cell-types of interest expressing specifically Green Fluorescent Protein (GFP) were isolated from the rest of the root using fluorescence-activated cell sorting (FACS).The outer layer of the root was found to be responding more than the inner part of the root after 48 hours of salt stress, with an overall down-regulation in both rice and Arabidopsis. Arabidopsis cortical cells responding to salt seem to regulate the cell wall biosynthesis, which may modulate the shape of the cells or alter the apoplastic movements of solutes in response to salt. Genes related to transport were affected by salt in Arabidopsis, with the crucial role of cortical cells in the movement of solutes being evident. Rice cortical cells respond to salt by showing a more extreme defence reaction in changing the protein metabolism and the regulation of transcription. The response of the inner part of the rice root to 48 hours of mild salt stress showed up-regulation of genes implicated in broader functional categories. The biological relevance of genes revealed using cell-type specific transcriptomics was demonstrated in a salt assay using knock-out (KO) lines of candidate genes from both cell-types in Arabidopsis thaliana. Three KO mutant lines showed 20% reduction in shoot sodium after 5 weeks of salt stress and were also able to maintain a higher shoot dry weight. These transcriptomic studies of isolated stelar and cortical cells in response to a mild salt stress have revealed salt responsive genes and pathways, indicating new areas for further study, and contributing to our understanding of the complex responses of plants to their environment at the cellular level.

Doctor of Philosophy / Entomology / Yoonseong Park / Tick salivary secretion is crucial for survival and for successful feeding. Tick saliva includes excretory water/ions and bioactive components for compromising the hosts' immune responses, and provides a direct route for pathogen transmission. Control of the tick salivation involves autocrine/paracrine dopamine, the most potent stimulator of tick salivation. Our research group reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Dopamine-induced salivary secretion was orchestrated by two distinct physiological roles via activation of the two dopamine receptors (Chapter 2). Low concentration of dopamine activated D1 receptor on epithelial cells of salivary gland acini leading inward fluid transport. High concentration of dopamine activated InvD1L receptors on axonal projections innervating myoepithelial cells modulating pumping/gating actions for emptying luminal saliva into the main duct. Thus, ticks coordinated salivary secretion with duo dopamine receptors. Dopamine-mediated saliva production involves an important downstream component, Na/K-ATPase (Chapter 3). Na/K-ATPase was found in the epithelial cells of all types of acini. However, Na/K-ATPase had two different functions in salivary secretion in different acini: 1) dopamine-mediated production of primary saliva in distally located salivary gland acini type-2/- 3, and 2) dopamine-independent resorption in proximally located salivary gland acini type-1. Type-1 acini were also found to function in direct water absorption of off-host ticks, which could be a potential route for delivery of acaricides. Chapter 4 investigated the comparative transcriptomics of the lone star tick underlying the processes of pathogen acquisition. Differential expression analyses in pathogen-exposed ticks revealed a number of transcripts that are important in the tick-pathogen interaction. These included genes for tick immunity against pathogen and for modulation of tick physiology facilitating a pathogen’s invasion and proliferation. My study expanded the understanding of physiological mechanisms controlling tick salivation. In addition, transcriptomics of ticks in interaction with pathogen identified several genes that are relevant in vector/pathogen interactions. The knowledge obtained in my study will facilitate to the development of novel methods for the disruption of tick feeding and pathogen transmission.

The aim of this thesis is to in various ways explore protein expression in human normal tissue and in cancer and to apply that knowledge in biomarker discovery. In Paper I the prognostic significance of RNA-binding motif protein 3 (RBM3) is explored in malignant melanoma. To further evaluate the prognostic significance of RBM3 expression was assessed in 226 incident cases of malignant melanoma from the prospective populationbased cohort study Malmö Diet and Cancer Study using tissue microarray technique (TMA). RBM3 was shown to be down regulated in metastatic melanoma and high nuclear expression in the primary tumor was an independent marker of prolonged over all survival. As a tool to facilitate clinical biomarker studies the Human Protein Atlas has created a tissue dictionary as an introduction to human histology and histopathology. In Paper II this work is introduced. A cancer diagnosis can be a complex process with difficulties of establishing tumor type in localized disease or organ of origin in generalized disease. Immunohistochemically assisted diagnosis of cancer is common practice among pathologists where its application combined with known protein expression profiles of different cancer types, can strengthen or help dismiss a suspected diagnosis. In Paper III the diagnostic performance of 27 commonly used antibodies are tested in a predominantly metastatic, multicancer cohort using TMA technique. Overall these 27 diagnostic markers showed a low sensitivity and specificity for its intended use, highlighting the need for novel, more specific markers. Breast, ovarian, endometrial and ovarian cancers affect predominantly women. Differential diagnostics between these cancer types can be challenging. In Paper IV an algorithm, based on six different IHC markers, to differentiate between these cancer types is presented. A new diagnostic marker for breast cancer, namely ZAG is also introduced. In Paper V the transcriptomic landscape of the adrenal gland is explored by combining a transcriptomic approach with a immunohistochemistry based proteomic approach. In the adrenal gland we were able to detect 253 genes with an elevated pattern of expression in the adrenal gland, as compared to 31 other normal human tissue types analyzed. This combination of a transcriptomic and immunohistochemical approach provides a foundation for a deeper understanding of the adrenal glands function and physiology.

Transcriptomics is the large-scale study of RNA molecules produced by the genome, in single cells or population of cells using high-throughput methods. With the advances in transcriptomic analysis, the monitoring of genome-wide gene expression provides a powerful approach for determining the action of drugs. In this study, we analyzed the transcriptional responses of cells treated with drugs either alone or in combinations to explore their effects in two different applications: breast cancer therapy and cell conversion. In the first part of this thesis, we aim at modeling the relationship between single and multidrug breast therapy at the transcriptome level. We monitored the effects of three drugs, and their combinations in human breast cancer MCF-7 cells using the cap analysis of gene expression method. We are the first to explore the impact of single and combinatorial treatment on promoter and enhancer expression on a genome-wide scale. After applying and customizing a broad spectrum of regression algorithms, we showed that the transcriptional response to combinatorial drug treatment at both promoters and enhancers is accurately described by a linear combination of the responses to the individual drugs. Our analysis is promising for eliciting the transcriptional reaction to multidrug therapies in an unbiased genome-wide way, which may minimize the need for exhaustive combinatorial screens. Following the drug combination analysis, we explored the possibility to systematically identify drugs that either alone or in combinations facilitate cell conversion. To date, no computational approach prioritizes or suggests chemical compounds promoting cell reprogramming. Using transcriptomic data of human primary cells and drug response expression profiles, we developed a computational framework which accurately predicts single drugs or drug cocktails driving any source cell type towards the desired lineage. Experimental and in-silico validation on human pluripotent stem cells confirms the ability of the top predicted drugs to enhance reprogramming. The introduced method has countless applications in regenerative medicine and can significantly speed up the research in this field.

Pour enrichir les connaissances scientifiques sur les effets biologiques des radionucléides et risques des contaminations chroniques sur la descendance, une étude multigénérationnelle in vivo d’exposition a été réalisée à doses non toxiques d'uranium. Ce modèle, a permis de suivre les effets biologiques de l’uranium sur trois générations de rats (F0, F1 et F2) par des analyses cliniques et le suivi de marqueurs biologiques. Dans cette étude, des analyses métabolomiques, transcriptomiques et épigénomiques ont été réalisées à partir d’échantillons de sang, d’urine et de rein.Pour la première génération des rats contaminés (F0), des différences dépendant du sexe des animaux sont observables par l’analyse des niveaux d’expression géniques (ARNm et micro-ARN) dans les reins, des profils métabolomiques et biochimiques dans les reins, l’urine et le sang. Aucune modification épigénétique des profils de méthylation de l’ADN rénal n’est à noter. Pour les deux générations suivantes (F1 et F2), un effet multigénérationnel dépendant aussi du sexe des rats est observable au niveau des profils métabolomiques urinaires et rénaux ainsi qu’au niveau des profils épigénétiques de méthylation de l'ADN des reins. Une baisse de poids corporel et des reins a aussi été observée pour la troisième génération de rats chez les mâles (F2).En conclusion, les travaux de cette thèse montrent qu’une contamination chronique à faible dose d'uranium entraine des effets biologiques sur plusieurs générations de rats. Ils sont observables à différents niveaux moléculaires des systèmes de régulation cellulaires et dépendent du sexe des rats. Ces effets, étroitement liés à des systèmes biologiques intégrés, sont utiles à la compréhension des mécanismes biologiques des expositions à l'uranium et à l’évaluation des risques de nocivités à long termes. Dans le domaine de la radioprotection, ces résultats justifient la nécessité de considérer les dimorphismes sexuels des individus et les conséquences des expositions sur les générations à venir. / In order to deepen scientific knowledge regarding biological effects of radionuclides and associated risk to offspring, an in vivo multigenerational study of chronic exposure to a non-toxic dose of uranium was performed by monitoring three generation of rats (F0, F1 and F2). Clinical parameters and biological markers, including metabolomics, transcriptomics and epigenomics high throughput analysis were conducted in blood, urine and kidney samples.For the first generation of contaminated rats (F0) sex-differences to uranium effects were observed in kidney for gene expression (mRNA, miRNA) and in kidney, urine and blood for biochemical parameters and metabolomics profiles. No epigenetic modification of DNA methylation profiles was shown in kidney. For the next two generations (F1, F2), a multigenerational sex-specific effect is observed for both metabolomics and renal DNA methylation profiles of contaminated rats. Moreover, for the last generation of male rats (F2), a decrease of both total body and kidney weight was shown.In conclusion, low-dose chronic contamination of rats to uranium leads to multigenerational effects. Including sex-differences, they can be shown at different molecular levels of the cellular system. Depending of integrated system biology, data of this thesis are useful in the understanding of biological mechanisms of uranium effect and risk of delayed harmful effect. In the field of radiation protection, these results prove the requirement of considering sexual dimorphisms and consequences of such exposures to offspring.

Thousands of chemical pollutants enter the environment continuously, each with the potential to cause adverse effects in both terrestrial and aquatic organisms. As a result, organisms are often exposed to a mixture of stressors within their habitat. Populations of fish inhabiting most aquatic environments are exposed to time-varying or repeated pulses of exposure, driven by run-off events or spills, or due to their mobility between polluted and clean waters. Therefore, the sustainability of fish populations is critically dependent on their ability to adapt to frequent changes in their local environment. Despite this, legislation to protect the environment from chemical contamination are generally based on toxicological measurements following exposures to single stressors, conducted under optimal laboratory conditions, and that do not take into account the variation in susceptibility of wild populations, or the potential consequences of exposure for the susceptibility of the population during future exposures, including across generations. Increasing evidence is suggesting that a number of chemicals may interact with the epigenome, and that differential responses to pollutants may be modulated, at least in part, via epigenetic mechanisms. However, our understanding of the role of epigenetic mechanisms in normal development in fish models or its susceptibility to exposure to environmental stressors is currently very limited. This thesis aimed to document the mechanisms of genetic and epigenetic responses to industrial pollutants in fish, and to explore the extent to which differential responses can be induced in the lab following exposure during the critical window of embryonic development or in adults. To address these objectives, I performed a series of experiments using both the zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus) as fish models. I first used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. I provide evidence of the sex-specific transcription of genes involved in reproduction and their regulation by epigenetic signalling in this commonly used vertebrate model and highlight important considerations regarding the use of whole tissues comprised of multiple cell types in epigenetic and transcriptomic studies. I then investigated the potential for exposure to Bisphenol A (BPA) to cause adverse effects on reproduction and to disrupt the expression profiles and promotor DNA methylation of target genes important for reproductive function and epigenetic signalling in the zebrafish. To do this, I exposed breeding zebrafish to a range of BPA concentrations over 15 days and found that BPA disrupted reproductive processes in zebrafish, likely via estrogenic mechanisms, but only at high concentrations. Importantly, exposure to environmentally relevant concentrations of BPA resulted in altered transcription of key enzymes involved in DNA methylation maintenance, and caused changes in promoter DNA methylation. I also conducted a series of repeated exposures to copper in the three-spined stickleback to investigate the extent to which differential susceptibility can be induced in the lab. This work provides evidence that pre-exposure to copper results in differential responses in future exposure scenarios both when the initial exposure occurred in adults and during embryogenesis. For adults, fish appeared to recover completely from the initial exposure following a period of depuration of 30 days, but displayed decreased susceptibility upon re-exposure. In contrast, for fish exposed during the critical windows of embryonic development when epigenetic reprogramming are hypothesised to occur, differential copper accumulation was maintained throughout life. Importantly, the initial exposure caused increased tolerance in the offspring, which was inherited up to the F2 generation. This work provides valuable information regarding potential critical windows of development which may be more susceptible to effects associated with pre-exposure, highlighting that early life exposure to a low concentration of copper can induce differential responses to copper across generations. These data highlight the extent of differential responses to chemical stressors likely to be present in wild populations, and point towards the possibility that effective population management will likely require an in-depth understanding of the exposure history of a given population in order to manage restocking initiatives, and to inform conclusions drawn from toxicity testing studies conducted using individuals originating from wild populations. In addition, these data suggest that it is likely that both epigenetic and genetic changes can contribute to the adaptation of individual populations to their local environment. Finally, other vertebrates including humans have been shown to be exposed to the chemicals tested in this thesis. Therefore, this highlights the potential for these chemicals to also cause toxic effects in humans, potentially via (epi) genetic mechanisms, and advocate the testing of the potential for inheritable phenotypes, such as those described in this thesis, to occur in mammalian models.

Sex and meiosis are ubiquitous in eukaryotes as the primary mode of reproduction. This suggests that despite the theoretical energetic advantages of asexual reproduction, organisms capable of sexual reproduction are at a much greater long-term evolutionary advantage. Rotifers, a group of microinvertebrates, offer unique opportunities to examine the evolution of sex due to their extensive proliferation, successful adaptation to a wide variety of ecological niches, and the diversity of reproductive modes represented in the group. The cyclically parthenogenetic monogonont rotifers have overcome constraints on the loss of sexual reproduction in order to frequently transition between sexual and asexual generations, making them a powerful system with which to address the maintenance of sex in animals. Obligately asexual bdelloid rotifers appear to have thrived without sex for tens of millions of years, a period of time much longer than expected given the hypothesized advantages of sexual reproduction. However, the molecular nature of sex and parthenogenesis is poorly understood in any rotifer species. To expand our knowledge of the molecular mechanisms of monogonont reproduction, we sequenced genomes of two distantly related species, Brachionus calyciflorus and Brachionus manjavacas and identified over 80 homologs for genes involved in meiotic processes. Several of these genes have undergone duplication events specific to the monogonont lineage, including genes with known roles in regulation of cell cycle transitions during meiosis. In addition, global gene expression patterns were determined using obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of B. calyciflorus. Quantitative comparison of expression between these strains revealed differentially expressed genes specific to sexual and asexual reproduction in this species, including genes related to dormancy/resting egg formation, meiosis, and hormone signaling pathways that are thought to be involved in the induction of sexual reproduction in monogononts. Finally, we analyzed gene expression in bdelloid rotifers for evidence of sexual reproduction or the utilization of meiotic genes under conditions inducing high levels of recombination. Through this work, we have established molecular markers for sexuality and asexuality in monogonont rotifers, and used these markers to evaluate reproduction in bdelloids. The data generated specifically allows for more informed analyses of the evolution of cyclical parthenogenesis and rotifer reproduction. Furthermore, this work extends the use of monogononts as a model system for addressing broader questions regarding the evolution of sexual reproduction.