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Long noncoding RNAs (lncRNAs) are being realized as important players in gene regulation and their misregulation has been considered as one of the underlying causes for tumor initiation and progression in many human pathologies. In the current thesis, I have addressed the functional role of lncRNAs in development and disease model systems. Genomic imprinting is an epigenetic phenomenon by which subset of genes are expressed in a parent of origin-specific manner. The Kcnq1 imprinted locus is epigenetically regulated by Kcnq1ot1 lncRNA. Deletion of an 890bp region at the 5’ end of Kcnq1ot1 in mouse resulted in the loss of silencing of neighboring ubiqui-tously imprinted genes (UIGs). In addition, we observed loss of DNA methylation at the UIG promoters. We have shown that Kcnq1ot1 RNA establishes CpG methylation by interacting with DNMT1. To explore the stability of lncRNA mediated silencing pathways, we have conditionally deleted Kcnq1ot1 in the mouse in a stage and tissue-specific manner. We have shown that Kcnq1ot1 is continuously required for maintaining the silencing of UIGs, whereas the silencing of the placental im-printed genes is maintained in an RNA independent manner.   To identify chromatin-associated lncRNA (CARs) on a genome-wide scale, we purified RNA from the sucrose gradient fractionated chromatin and subjected it to RNA sequencing. Our study has identified 141 intronic and 74 long intergenic CARs. Characterization of one of the CARs revealed that it regulates the expression of neighboring genes in cis by modulating the chromatin structure.   We have explored the functional role of lncRNA in tumor progression and initiation by using pediatric neuroblastoma. By transcriptional profiling of low- and high-risk tumors, we have identified several lncRNAs differentially expressed between these subtypes. We report an uncharacterized RNA NBAT-1, expressed at lower levels in high-risk tumors relative to low-risk tumors.  Using neuroblastoma cell culture system, we demonstrated that NBAT-1 has anti-cell proliferative and anti-invasive properties. In addition, it promotes differentiation of neurons from undifferentiated neuroblastoma cell lines.   In summary, by employing mouse genetics, cell culture based model system and expression profiling in tumors, we have uncovered new roles of lncRNA in gene regulation.

The main thrust of this thesis was to characterize P.falciparum genetic diversity in northern Ghana. To do this, I used simple techniques to purify P. falciparum DNA from clinical samples across a rural setting for whole-genome sequencing. The goal was to provide a framework for exploring host-parasite genetic interactions. Utilizing Illumina deep sequencing data for 277 isolates I analyzed P. falciparum genetic diversity and described within-host diversity across this area. I observed random mating (ie no population structure) in the local parasite population, and a high genetic diversity indicative of high out-crossing. Moreover, when I aggregated my data with similar published data from Burkina Faso and Mali (sites ≈500km apart), no population structure was evident. In contrast, sites sampled in Cambodia and Thailand (≈ 800km apart) were found to have greater population structure and high potential for inbreeding. This may be driven by differences in transmission intensity between the sites sampled in West Africa and southeast Asia. To demonstrate the utility of deep sequencing data, I focused on the genomic regions of pfdhfr, pfdhps and pfcrt, known to be under antimalarial drug selection. I surveyed the full diversity of point mutations already characterized in these genes and discovered previously unknown variants. However, in order to provide a means to follow up on new variants or interesting candidate regions in large clinical samples with limited parasite DNA, I assessed the Sequenom iPLEX platform for high-throughput genotyping of P. falciparum polymorphisms. This necessitated developing a method appropriate for assigning genotypes in haploid genome mixtures common in natural infections. Finally, I used this method to type host and parasite markers in a case-control sample set from this region for exploring host-parasite genetic interactions. I found that children who have the sickle-cell trait and carry parasites that have pfdhfr resistant alleles lose their protection against severe malaria as compared to children who have normal haemoglobin and are infected with parasites with these resistant alleles.

Les recherches sur les ARN non codants et leurs rôles biologiques ont transformé la conception des propriétés des génomes et de la régulation de l'expression génétique. Ces recherches sont ici analysées dans une perspective historique et philosophique. Elles permettent de caractériser certaines transformations théoriques, conceptuelles et méthodologiques affectant la biologie moléculaire contemporaine. La diversité des démarches d'investigation employées dans les travaux sur les ARN non codants est soulignée. Parmi ces démarches, les expériences exploratoires occupent une place importante, et sont à l'origine de bouleversements conceptuels et théoriques majeurs. L'affirmation selon laquelle la découverte des ARN non codants constitue une révolution scientifique est nuancée, et les apports de cette découverte sont caractérisés dans toute leur diversité. Les limites rencontrées par le concept de gène dans la biologie post-génomique sont précisées, et reliées à l'histoire de ce concept. Un statut possible pour le gène est alors proposé ; il reconnaît la diversité des éléments génétiques fonctionnels, la dimension historique des génomes, et les différents niveaux d'étude de la relation entre génotype et phénotype. Les recherches sur les ARN non codants affaiblissent par ailleurs la pertinence théorique du discours informationnel pour décrire la diversité des fonctions assurées par les acides nucléiques. Enfin, ce travail montre que les explications du rôle régulateur des ARN mettent en jeu différents types explicatifs, dépassant le cadre mécaniste. Un pluralisme explicatif est défendu, et le statut du concept de mécanisme dans la biologie moléculaire contemporaine est clarifié. / Research on non-coding RNAs and their biological roles has transformed the way biologists conceptualize genomic properties and the regulation of genetic expression. This work analyzes this research from both a historical and philosophical perspective. It helps characterize some theoretical, conceptual and methodological transformation currently affecting moleculau biology. I stress that multiple modes of investigation are employed in research on non coding RNAs. Particularly important among them is exploratory experimentation, which brings about major conceptual and theoretical changes. I temper the claim that the discovery of non-coding RNAs has triggered a scientific revolution and characterize the multifaceted contributions of this discovery. I identify some limits faced by the gene concept in post-genomic biology, and relate them to the history of this concept. I suggest a possible status for the gene, which to study the relation between genotype and phenotype. Besides, research on non-coding RNAs leads to question the theoretical relevance of informational concepts to describe the diverse functions fulfilled by nucleic acids. Finally, I show that explanations of the regulatory roles of RNAs involve different explanatory schemes, which do not fit the mechanistic framework. I advocate an explanatory pluralism, and clarify the status of the concept of mechanism in contemporary molecular biology.

A hibridação pode ter várias consequências, incluindo a manutenção ou aumento da diversidade resultando em zonas de hibridação estáveis; a introdução de nova variedade alélica que pode acarretar na origem ou transferência de adaptações, que pode ter efeito positivo no resgate de populações pequenas puras; o reforço do isolamento reprodutivo e a formação de novas linhagens híbridas. Além disso, a hibridação pode reduzir a diversidade, pois a quebra das barreiras reprodutivas e a mescla de populações anteriormente distintas pode acarretar na extinção de uma das populações ou espécies envolvidas na hibridação. Nesse cenário, nosso estudo teve como objetivo estudar uma zona de hibridação no estado da Bahia entre duas espécies de aves, Pyriglena atra (Swaison 1825) e P. leucoptera (Vieillot 1818). Foram analisados mais de 20.000 SNPs de 20 indivíduos de P. leucoptera, 22 de P. atra e seis indivíduos intermediários morfológicos. Encontramos pouca a nenhuma mistura genética nos intermediários morfológicos, o que indica um descompasso entre genótipo e fenótipo, o que poderia estar associado ao fato de os marcadores genéticos analisados terem.se mostrado neutros, enquanto a morfologia externa pode estar sob pressão de seleção sexual. Foi detectado que as localidades adjacentes à zona de hibridação apresentaram mistura genética na maioria dos indivíduos, um indicativo de que além de hibridação, esteja ocorrendo introgressão entre as espécies, sendo maior em P. leucoptera, o que pode ser consequência de \"genetic surfing\" / Hybridization can have many consequences, including maintenance or increase of diversity resulting in stable hybridization zones; introduction of a new allelic variety that can lead to the origin or transfer of adaptations, that may result on a positive effect on the rescue of small pure populations; enhancement of reproductive isolation and the origin of new species. In addition, hybridization may reduce diversity, as the break of reproductive barriers and the mixture of previously distinct populations may lead to the extinction of one of the populations or species. In this scenario, our study aimed to study a hybridization zone in the state of Bahia between two bird species, Pyriglena atra (Swaison 1825) and P. leucoptera (Vieillot 1818). We analyzed more than 20,000 SNPs from 20 individuals of P. leucoptera, 22 P. atra and six morphological intermediate individuals. We found little or no genetic mixture in the morphological intermediates, which indicates a mismatch between genotype and phenotype, possibly because these molecular markers were tested as neutral and these morphological characters could be under sexual selection. The majority of the individuals from localities adjacent to the hybrid zone have mixed ancestry, an indication that, besides hybridization, introgression among the species is occurring but higher into P. leucoptera as a possible consequence of genetic surfing

Genomic instability and aneuploidy, which are ubiquitous hallmarks of cancer cells, encompass both structural and numerical chromosome aberrations. Strikingly, cancer cells often display recurrent patterns of aneuploidy which are thought to be contingent on selection pressures within the tumour microenvironment maintaining advantageous karyotypes. However, it is currently unknown if individual chromosomes are intrinsically vulnerable to missegregation, and therefore whether chromosome bias may also contribute to pathological aneuploidy patterns. Moreover, the earliest responses to chromosome missegregation in non-transformed cells, and how these are overcome in cancer, has remained elusive due to the difficult nature of isolating nascent aneuploid cells. Results. Individual chromosomes displayed recurrent patterns of biased missegregation in response to a variety of cellular stresses across cell lines. Likewise, a small subset of chromosomes accounted for a large fraction of segregation errors following one specific mechanism driving aneuploidy. This was supported by the discovery that chromosomes 1 and 2 are strikingly susceptible to the premature loss of sister chromatid cohesion during prolonged prometaphase arrest. Additionally, I have elucidated the arrangement of individual metaphase human chromosomes, highlighting missegregation vulnerabilities occurring at the metaphase plate periphery following nocodazole wash-out. Finally, I have developed a novel system for isolating nascent aneuploid cells, suggesting the earliest transcriptome responses to chromosome missegregation in non-transformed human cells involve ATM and BCL2-mediated apoptosis.

To fulfil global energy demand and to mitigate economical, geopolitical and ecological challenges associated with fossil fuel utilisation, the energy sector is moving towards greater use of sustainable and environmentally friendly energy sources, including biofuels. The ideal transport biofuel would be hydrocarbons that are identical to fossil petroleum. However, to date characterised hydrocarbon biosynthetic pathways include a decarbonylation or decarboxylation reaction, which involves the loss of one carbon resulting in odd-numbered carbon chain hydrocarbons. This carbon loss decreases carbon efficiency for alkane production, which reduces microbial fuel economic competitiveness. Therefore, it is key that new pathways for alkane production are identified. The sulphate-reducing bacteria genus Desulfovibrio was previously reported to synthesise even-numbered carbon chain alkanes, which suggests an alternative route for alkane production without carbon loss. This investigation aimed to verify Desulfovibrio alkane biosynthesis and characterise the possible synthetic pathway. Ten Desulfovibrio strains, representing seven species, were screened for alkane synthesis using isotopically labelled growth media. The ability to produce alkanes within the Desulfovibrio genus was confirmed and was shown to be strain-specific under a set of culture conditions. The biogenic alkanes detected were octadecane (C18), nonadecane (C19) and eicosane (C20), with a predominance of even-numbered carbon chain alkanes. Fatty acid analysis of Desulfovibrio strains showed an alkane biosynthetic pathway was unlikely to involve a decarbonylation or decarboxylation step. A novel hypothesis was therefore proposed that alkane biosynthesis by Desulfovibrio follows a metabolic route, which has not previously been characterised, involving a series of reduction reactions from the fatty acid pool. The characterisation of the putative Desulfovibrio hydrogenation pathway for alkane biosynthesis was undertaken via a target-directed genome mining approach. The genomic DNA of nine Desulfovibrio spp. was purified, sequenced, de novo assembled and annotated. Seven of these nine genomes are unpublished to date. No homologs of previously characterised alkane biosynthetic enzymes from bacteria were in silico identified in the genomes and proteomes of alkane producing Desulfovibrio spp., suggesting that Desulfovibrio alkane biosynthetic pathway is likely to be catalysed by currently uncharacterised enzymes. The 16S rRNA-based phylogeny of Desulfovibrio spp. supported the hypothesis that the Desulfovibrio alkane biosynthetic pathway was acquired by a common ancestral strain via horizontal gene transfer. The ability of Desulfovibrio to produce alkanes was therefore hypothesised to be due to the presence of recruited genes encoding enzymes involved in alkane synthesis. A comparative genomic analysis intersecting six-alkane producing and four non-alkane producing Desulfovibrio genomes resulted in the in silico identification of 33 hypothetical proteins considered with high confidence to be exclusive to alkane producing Desulfovibrio strains. A novel hypothetical Desulfovibrio alkane biosynthetic pathway was proposed involving a V-type ATPase, an uncharacterised protein, named as a putative reductase in this investigation, and a putative methyltransferase, which were predicted to be exclusive to alkane producing Desulfovibrio spp. The inorganic phosphates resulting from the ATP hydrolysis catalysed by the V-type ATPase would be involved in a reaction with fatty alcohols to form alkyl phosphates, which are putative activated intermediates required for the hydrogenation route from fatty alcohols to alkanes. The putative reductase and the methyltransferase, predicted to share similar structural features with known alkane-binding proteins, would subsequently reduce alkyl phosphates to alkanes and to iso-alkanes respectively. Empirical investigation of the candidate molecular basis function in Desulfovibrio alkane biosynthesis was undertaken. The Desulfovibrio alkane biosynthetic pathway remains to be fully characterised.

The management of genomic diversity is important within breeding programs and is primarily achieved through controlling the rate of inbreeding. A failure to adequately manage the rate of inbreeding will result in an increased risk of the expression of lethal recessive mutations, inbreeding depression and losses in genetic variance, thereby restricting long-term genetic progress. Each research chapter within this thesis used real data collected from a commercial pig breeding operation to examine a key area of research regarding the management of genomic diversity. The first research chapter examined the selection outcomes from the practical application of Optimal Contributions (OC). These outcomes were examined to determine their alignment with the current theories regarding selection, particularly as to the extent by which selection decisions were influenced by estimated Mendelian sampling terms. This assessment was conducted for the initial selection of individuals as parents, which parents went on to provide a long-term contribution and the magnitude of these contributions. OC was shown to have shifted breeding decisions more closely in alignment with the estimated Mendelian sampling terms. The second research chapter used genomic data to assess the adequacy of the pedigree-based approach for managing diversity during selection. This approach assumes the infinitesimal model with all loci neutral and no impact from selection per se on heterozygosity. Using genomic information, the observed loss of heterozygosity at each marker was compared to the loss of heterozygosity expected from the pedigree-based relationships. Regional disparities between the observed and expected losses in heterozygosity were detected, which were potentially attributable to selection. Runs of homozygosity and the pairwise linkage disequilibrium between markers were also examined within these regions. Regions showing disparity were found to contain well validated quantitative trait loci for important traits. The third research chapter sought to provide a genomic solution to the shortcomings of the pedigree-based approach for quantifying relatedness, identified above. A methodology was devised for tracing identity by descent (IBD) at each allelic position over five ancestral generations, following phasing and imputation of the genomic data. A comparison was made between the inbreeding expected from the pedigree relationships and that observed from the identity by descent of genomic information. In the population studied it was not currently feasible to derive a relationship matrix based exclusively on observed IBD. The fourth research chapter used imputed genomic information to identify haplotypes which had a putative lethal recessive effect. Haplotypes which were never observed in the homozygous form, either in the population or in the offspring produced between carriers, were classified as candidate haplotypes. The top candidates on each chromosome were then examined for a reduction in the total number born when two carriers were mated together. A total of six putative lethal recessive haplotypes were detected relating to at least four putative lethal recessive mutations, where one homozygote was absent and the size of the reduction in litter size matched that expected for a lethal recessive effect. The research chapters contained within this thesis demonstrate the important role that genomics can have in managing inbreeding in addition to generating genetic gain. Genomics is able to provide a more accurate prediction of the Mendelian sampling term, better quantify the relatedness between individuals and detect lethal recessive effects.