Filariasis and onchocerciasis are debilitating diseases affecting 120 million people globally. The massive socio-economic impact of these diseases energized the international community to declare a goal of eliminating filariasis 2020. This resulted in a dramatic increase in the efforts to eliminate filariasis and onchocerciasis, employing a strategy of mass drug administration (MDA). However, these programs rely upon the small arsenal of drugs. This leaves these programs vulnerable to failure in the face of developing resistance and local intolerance to the current drug regimens. Thus, new drugs against these infections are critically needed. A homologue of the ecdysone receptor (EcR), a master regulator of development in insects, has been identified in B. malayi. The potential of the EcR as a drug target has been underscored by work in the agricultural industry, where insecticides targeting the ecdysone developmental pathway are effective and non-toxic to non-target species. As the EcR is absent in humans, it represents an attractive potential chemotherapeutic target. The first study investigates the hypothesis that the ecdysone receptor controls the embryogenesis and molting in the filarial parasite. In-vitro embryogram and in-vivo phenotypic studies were conducted to delineate the effect of 20-hydroxyecdysone on the Brugia malayi parasites. The results suggest that the hormone accelerates embryogenesis and causes precocious molts, resulting in the death of the parasite. Further, transcriptomic and proteomic analysis of the ecdysone treated worms provided evidence that the up-regulated genes participate in embryogenesis. Based upon the validation of the ecdysone receptor as a potential drug target, subsequent studies focused on the development of a drug discovery model to screen for agonists and antagonists of the B. malayi ecdysone receptor. A stable cell line was created to aid the high throughput screening to rapidly identity agonist and antagonist compounds. A total of 7 agonists and 2 antagonists were identified. A homology model of the BmEcR ligand-binding domain was created as an alternate method for virtual screening of small molecules as well as to study the ligand-receptor interactions. The hits identified with the assay were docked in the active site of the BmEcR homology model providing an excellent correspondence of data between the molecular assay and the virtual screening method.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Amburana cearensis (cumaru) Ã uma leguminosa subutilizada da Caatinga. O alto teor proteico das sementes faz delas uma possÃvel nova fonte de proteÃnas alimentares. Foram determinadas as melhores condiÃÃes de extraÃÃo proteica para posterior produÃÃo do isolado proteico de cumaru (IPAc). As anÃlises tambÃm foram conduzidas com a farinha delipidada (FDAc) e com um isolado proteico de soja comercial (IPS). IPAc apresentou teor proteico > 90% e composiÃÃo de aminoÃcidos comparÃvel a IPS, com notÃvel deficiÃncia em metionina. Foram determinadas algumas propriedades funcionais para predizer sua adequaÃÃo como ingrediente na indÃstria alimentÃcia. Os resultados para FDAc, IPAc e IPS foram, respectivamente: solubilidade 20-79; 9-99 e 3-76%; capacidade de retenÃÃo de Ãgua 1,7; 3,9 e 7,5 g/g; capacidade de retenÃÃo de Ãleo 1,5; 6,3 e 1,6 g/g; capacidade emulsificante / estabilidade 52 / 46%; 55 / 51% e 53 / 21%; capacidade espumante / estabilidade 47 / 32%; 65 / 53% e 33 / 8 e menor concentraÃÃo geleificante 18, 16 e 12%. Para investigar se IPAc e IPS apresentam indÃcios de toxicidade, cÃlulas de adenocarcinoma humano foram expostas por 24 h aos isolados. Para comparaÃÃo, tambÃm foi realizada exposiÃÃo com os isolados de soja (IPGm), feijÃo comum (IPPv), feijÃo de porco (IPCe) e mamona (IPRc), a fraÃÃo proteica de mamona (FPRc), as lectinas PHA-E e ConA e o controle quÃmico (Crtl_Quim). O RNA das cÃlulas foi hibridizado em microarranjos contendo o genoma humano completo. Os perfis de expressÃo gÃnica apÃs exposiÃÃo Ãs proteÃnas foram comparados aos de PBS e Crtl_Quim e agrupados hierarquicamente. Crtl_Quim, IPAc e IPGm se mostraram inadequados para a continuaÃÃo das anÃlises por provocar um efeito superestimado. A anÃlise de vias e processos biolÃgicos afetados apontou que genes envolvidos com a biossÃntese de colesterol, estresse do RE e resposta imune foram superexpressos e genes envolvidos com o ciclo celular e replicaÃÃo do DNA foram subexpressos apÃs exposiÃÃo a IPPv, IPCe, PHA-E e ConA. As amostras IPRc e FPRc regularam para cima genes envolvidos com a apoptose e resposta imune e para baixo genes envolvidos com o ciclo celular e sinalizaÃÃo do cAMP. AnÃlise do mapa conectivo mostrou alta correlaÃÃo de ConA, PHA-E e IPCe com drogas bloqueadoras de canais de Ca2+ e de K+; enquanto IPRc e FPRc apresentam funÃÃo biolÃgica semelhante a pelo menos seis drogas inibitÃrias da sÃntese proteica. Conclui-se que IPAc tem grande potencialidade como novo alimento proteico com grande aplicabilidade na indÃstria de alimentos, devendo, no entanto, sofrer modificaÃÃes no mÃtodo de obtenÃÃo visando a melhora na solubilidade e a eliminaÃÃo do sal residual. / Amburana cearensis (cumaru) is an underutilized legume from Caatinga. Because of their high protein content its seeds are a possible novel source of food protein. Were determined the best conditions for protein extraction and preparation of protein isolate (IPAc).Analysis were carried out also with the defatted flour (FDAc) and with a marketed soybean protein isolate (IPS). IPAc showed protein content of 90% and amino acid composition compatible with the IPSâs composition, with remarkable low methionine content. Some functional properties were measured in order to predict the adequacy of the isolates as an ingredient for food industry. The results for FDAc, IPAc and IPS were, respectively: solubility 20-79; 9-99 e 3-76%; water holding capacity 1.7; 3.9 e 7.5 g/g; oil holding capacity 1.5; 6.3 e 1.6 g/g; emulsion formation / stability 52 / 46%; 55 / 51% e 53 / 21%; foamability / stability 47 / 32%; 65 / 53% e 33 / 8; least gelling concentration (LGC) 18, 16 and 12%. In order to investigate whether IPAc e IPS exhibit signs of toxicity, two cell lines of human adenocarcinoma (MCF-7 e Caco-2) were exposed to these isolates as well as to others prepared from soyabean (IPGm), white bean (IPPv), Jack bean (IPCe) and castor bean (IPRc), to the protein fraction from castor bean (FPRc), to the lectins PHA-E and ConA (50 Âg/mL) and to the chemical control (Crtl_Quim). Subcitotoxic doses were determined by viability test to later exposure assay for 24 h. The MCF-7 cellâs RNA were extracted and hybridized to a microarray containing the complete human genome. The expression profiles after the exposition to the proteins were compared to that ones generated after PBS and Crtl_Quim and hierarchicaly clustered. Crtl_Quim, IPAc and IPGm were unsuitable for further analysis by their overestimated effect. Analysis of biological pathways and process showed that genes involved with the cholesterol biosynthesis, ER stress and immune response were upregulated and genes involved with cell cycle and DNA replication were downregulated after exposure of cells to IPPv, IPCe, PHA-E and ConA. The samples IPRc and FPRc upregulated genes involved with apoptosis and immune response and downregulated genes involved with cell cycle and cAMP signaling. Connectivity Map analysis showed high correlation of ConA, PHA-E and IPCe with drugs that are Ca2+ and K+ channel blockers, while IPRc and FPRc showed biological function similar to at least six drugs inhibitors of protein synthesis. Itâs possible to conclude that the IPAc has a high potentiality as a novel protein food with wide applicability in the food industry and should, however, be modified in the method of obtaining aiming the improvement in its solubility and eliminating the residual salt.

Precision medicine provides targeted treatment for an individual patient based on disease mechanisms, promoting health care. Matched transcriptomes derived from a single subject enable uncovering patient-specific dynamic changes associated with disease status. Yet, conventional statistical methodologies remain largely unavailable for single-subject transcriptome analysis due to the "single-observation" challenge. We hypothesize that, with statistical learning approaches and large-scale inferences, one can learn useful information from single-subject transcriptome data by identifying differentially expressed genes (DEG) / pathways (DEP) between two transcriptomes of an individual. This dissertation is an ensemble of my research work in single-subject transcriptome analytics, including three projects with varying focuses. The first project describes a two-step approach to identify DEPs by employing a parametric Gaussian mixture model followed by Fisher's exact tests. The second project relaxes the parametric assumption and develops a nonparametric algorithm based on k-means, which is more flexible and robust. The third project proposes a novel variance stabilizing framework to transform raw gene counts before identifying DEGs, and the transformation strategically by-passes the challenge of variance estimation in single-subject transcriptome analysis. In this dissertation, I present the main statistical methods and computational algorithms for all the three projects, as well as their real-data applications to personalized treatments.

Les études d'association pangénomiques (GWAS) ont permis l'identification de plusieurs dizaines de gènes et de polymorphismes nucléotidiques (SNPs) contribuant au risque de diabète de type 2.Plus généralement, les GWAS ont permis d'identifier des milliers de SNPs contribuant à des maladies complexes chez l'Homme.Cependant, la caractérisation fonctionnelle et les mécanismes biologiques impliquant ces SNPs et ces gènes restent en grande partie à explorer.En effet, les conséquences de ces polymorphismes sont complexes et peu connues. Une conséquence directe est l'altération de la protéine codée par un gène, voire une extinction complète de la transcription du gène (p.ex. via l’introduction d’un codon stop dans la séquence).Par ailleurs, ces polymorphismes peuvent avoir un rôle de régulation dans l'expression des gènes, par exemple, en perturbant la liaison de facteurs de transcription et d'enzymes impliqués dans la méthylation de l'ADN.Malgré des associations fortes des SNPS identifiés, ils ne peuvent expliquer la totalité de l'héritabilité du diabète de type 2, suggérant par le fait même des mécanismes d'interactions entre les différentes couches que représentent la génomique, la transcriptomique et l'épigénomique.Le changement de paradigme en statistique génétique et la disponibilité de données transcriptomiques et épigénomiques sont responsables de l'évolution du domaine, passant des analyses d'associations à des analyses transversales de type multi-omique, et permettant de fournir des éléments de réponse sur l’aspect fonctionnel des SNPs ou des gènes impliqués, et dans certains cas, permettant d'évaluer le lien causal de ces variants sur la pathologie.Les développements et applications méthodologiques proposés dans cette thèse sont variés, allant d’une approche similaire aux GWAS, mettant à profit les données longitudinales disponibles dans certaines cohortes (p.ex. D.E.S.I.R.), au moyen d'un modèle joint; de la caractérisation fonctionnelle de gènes candidats, identifiés par GWAS, dans la sécrétion d'insuline par une étude transcriptomique multi tissu et dans un modèle cellulaire; de l'identification d'un nouveau gène candidat (PDGFA) impliqué dans la dérégulation de la voie de l'insuline dans le diabète de type 2 via des mécanismes épigénétiques et transcriptomiques; et enfin de la caractérisation de l'effet sur le transcriptome de deux substituts du bisphénol A dans un modèle d’adipocyte primaire.L'augmentation des connaissances des processus biologiques dans lesquels sont impliqués les SNPs et gènes identifiés par GWAS pourrait permettre l'élaboration de stratégies diagnostiques plus efficaces, ainsi que l'identification de cibles thérapeutiques pour le traitement du diabète de type 2 et des complications associées (p.ex. insulinorésistance, NAFLD, cancer, etc.). Plus généralement, ces études multi-omiques ouvrent la voie à l'approche émergente que représente la médecine de précision, permettant le traitement et la prévention des pathologies tout en prenant en compte ce qui fait la spécificité d'un individu, à savoir son génome et son environnement, tous deux interagissant sur son transcriptome et son épigénome. / Genome-wide association studies (GWAS) have resulted in the identification of several dozen of genes and single nucleotide polymorphisms (SNPs) contributing to type 2 diabetes.More generally, GWAS have identified thousands of SNPs contributing to complex diseases in humans.However, the functional characterization and biological mechanisms involving these SNPs and genes remain largely to be explored. Indeed, the consequences of these polymorphisms are complex and little known.One direct consequence of the SNPs is the alteration of the protein encoded by a gene, or even a complete transcriptional gene silencing (e.g. codon stop in the sequence). Furthermore, these polymorphisms may have a regulatory role in gene expression, for example, by interfering with the binding of transcription factors and enzymes involved in DNA methylation.Despite the strong associations of identified SNPs, they cannot explain the full heritability of type 2 diabetes, suggesting interactions mechanisms between the different layers of -omics, such as genomics, transcriptomics and Epigenomics.The shift of paradigm in statistical genetics and the availability of transcriptomic and epigenomic data are responsible for the evolution of the discipline, moving from association studies to multi-omics, and providing insights on the functional aspect of the SNPs or genes involved, and in some cases allowing to evaluate the causal link of these variants on the pathology.The methodological developments and their applications proposed in this thesis are various, ranging from a similar approach to GWAS, leveraging the longitudinal data available in some cohorts (e.g. D.E.S.I.R.), using an joint model approach; the functional characterisation of candidate genes in insulin secretion by a multi tissue transcriptomic study and transcriptomic study in a cell model; the identification of a new candidate gene (PDGFA) involved in the deregulation of the insulin\\\'s pathway in type 2 diabetes through epigenetic and transcriptomic mechanisms; and finally, the characterisation of the effect on the transcriptome of two substitutes of bisphenol A in a primary adipocyte model.The increase of knowledge in biological processes involving SNPs and genes identified by GWAS could enable the development of more effective diagnostic strategies, and the identification of therapeutic targets for the treatment of type 2 diabetes and associated complications (e.g., insulin resistance, NAFLD, cancer, etc.).More generally, these multi-omics studies pave the way for the emerging approach of precision medicine, allowing the treatment and prevention of pathologies while taking into account what makes the specificity of an individual, namely his genome and his environment, both interacting on his transcriptome and his epigenome.

In the teleost brain, radial glial cells (RGCs) are the main macroglia and are stem- like progenitors that express key steroidogenic enzymes, including the estrogen- synthesizing enzyme, aromatase B (cyp19a1b). As a result, RGCs are integral to neurogenesis and neurosteroidogenesis in the brain, however little is known about the permissive factors and signaling mechanisms that control these functions. The aim of this thesis is to investigate if the secretogranin-derived neuropeptide secretoneurin (SN) can exert regulatory control over goldfish (Carassius auratus) RGCs. Immunohistochemistry revealed a close neuroanatomical relationship between RGCs and soma of SNa- immunoreactive magnocellular and parvocellular neurons in the preoptic nucleus in both goldfish and zebrafish (Danio rerio) models. Both intracerebroventricular injections of SNa into the third brain ventricle and SNa exposures of cultured goldfish RGCs in vitro show that SNa can reduce cyp19a1b expression, thus implicating SNa in the control of neuroestrogen production. RNA-sequencing was used to characterize the in vitro transcriptomic responses elicited by 1000 nM SNa in RGCs. These data revealed that gene networks related to central nervous system function (neurogenesis, glial cell development, synaptic plasticity) and immune function (immune system activation, leukocyte function, macrophage response) were increased by SNa. A dose-response study using quantitative proteomics indicates a low 10 nM dose of SNa increased expression of proteins involved in cell growth, proliferation, and migration whereas higher doses down- regulated proteins involved in these processes, indicating SNa has dose-dependent regulatory effects. Together, through these altered gene and protein networks, this thesis proposes SNa exerts trophic and immunogenic effects in RGCs. These datasets identified a total of 12,180 and 1,363 unique transcripts and proteins, respectively, and demonstrated that RGCs express a diverse receptor and signaling molecule profile. Therefore, RGCs can respond to and synthesize an array of hormones, peptides, cytokines, and growth factors, revealing a multiplicity of new functions critical to neuronal-glial interactions.

Haemophilus influenzae (Hi) é uma bactéria Gram-negativa comensal da nasofaringe e um patógeno oportunista cujo único hospedeiro natural conhecido é o ser humano. As cepas de Hi que possuem cápsula de polissacarídeo estão associadas a doenças invasivas mais graves, sendo as de sorotipo b (Hib) as principais causadoras da meningite bacteriana em populações não vacinadas. Para produzir a vacina contra Hib, o polissacarídeo purificado desta bactéria é conjugado quimicamente ao toxóide tetânico. Industrialmente, a produção do polissacarídeo é realizada cultivando esse micro-organismo em biorreatores, entretanto o rendimento em polissacarídeo é baixo, mesmo com fornecimento de nutrientes, controle de pH e outros ajustes das condições no decorrer do cultivo. O estudo dos diferentes perfis fisiológicos da população bacteriana de Hib no decorrer do cultivo através da transcritômica traz a possibilidade de aprofundar o conhecimento sobre o metabolismo desse micro- organismo. As taxas de transcrição dos genes expressos em diferentes momentos considerados como pontos metabolicamente significativos do cultivo de Hib linhagem GB 3291 em batelada alimentada conduzido em Biorreator de 10 L, com aeração submersa e controles de pH (7,0) e temperatura (30° C) foram obtidas através de sequenciamento de RNA paralelo massivo (RNA-seq). A análise de co-expressão dos genes foi realizada com WGCNA, em que oito módulos de genes co-expressos foram identificados, quatro dos quais apresentaram correlação alta com dados fenotípicos dos cultivos, inclusive produtividade de acetato e de polissacarídeo. Análise de enriquecimento funcional identificou vias metabólicas associadas a ribossomo, síntese de parede celular, transportadores e consumo de carbono. A análise de expressão diferencial permitiu observar o comportamento desta bactéria durante o cultivo. Através da análise das taxas de transcrição dos genes foi possível identificar as principais vias de síntese de acetato e de polissacarídeo capsular, sendo esta última feita principalmente através da via de pentose fosfato, em detrimento da via de interconversão pentose-glucuronato. Nossos dados mostram que as diferentes etapas do cultivo de Hib leva à ação conjunta de vários grupos de genes, com destaque àqueles ligados às funções celulares básicas, como a síntese de proteínas e de parede celular, o transporte e a síntese de aminoácidos. Esses resultados contribuem para o entendimento dos processos bioquímicos e celulares que ocorrem durante o processo de cultivo de Hib, possibilitando que sejam feitas sugestões de modificações genéticas em Hib e alteração no processo de cultivo com propósito de diminuir produção de acetato e aumentar produção do polissacarídeo. / Haemophilus influenzae (Hi) is a nasopharynx commensal Gram-negative bacterium and an opportunistic pathogen whose only known natural host is human being. Hi strains with polysaccharide capsule are related to more severe invasive diseases, wherein type b capsule (Hib) strains are the main cause of bacterial meningitis in unvaccinated population. To produce Hib vaccine, purified polysaccharide of this bacterium is chemically conjugated to tetanus toxoid protein. Industrially, polysaccharide production is performed by cultivating this micro-organism in bioreactors; however, the yield of polysaccharide is low, even with supply of nutrients, pH control and further adjustments of the fermentation conditions during cultivation. The study of different physiological profiles of Hib bacterial population during cultivation by transcriptomics brings the possibility to deepen the knowledge about the metabolism of this micro-organism. Transcription of genes expressed at different times considered metabolically significant points of Hib strain GB 3291 grown in fed-batch conducted in a 10 L bioreactor with submerged aeration and pH (7.0) and temperature (30 ° C) control rates were obtained through massive parallel RNA sequencing (RNA-seq). Gene co-expression analysis was performed with WGCNA, in which eight modules of co-expressed genes were identified, four of which showed high correlation with cultivation data traits, including acetate and polysaccharide productivity. Enrichment analysis identified pathways related to ribosome, cell wall synthesis, transports and carbon consumption. Differential expression analysis allowed to observe this bacteria behaviour during cultivation. Through transcription rate analysis, it was possible to identify the main pathways for acetate and polysaccharide synthesis, which is through pentose phosphate pathway instead of glucoronate-pentose pathway. Our data show that different stages in Hib cultivation leads to joint action of several gene groups, highlighting genes related to basic cellular roles, like protein and cell wall synthesis, transport and aminoacid synthesis. These results contribute to the understanding of biochemical and cellular processes that ocurr during Hib cultivation process, allowing suggestions to be made to modify Hib gene circuitry and to change cultivation process in order to decrease acetate production and to decrease acetate production and increase polysaccharide production.

L’infertilité masculine est actuellement considérée comme un problème majeur qui pose une situation alarmante sur la santé publique. L’oligozoospermie, l’asthénozoospermie et la tératozoospermie sont les trois anomalies les plus connues des spermatozoïdes. Elles affectent, respectivement, la densité, la motilité et la morphologie des spermatozoïdes. Un spermatozoïde anormal est très souvent corrélé à des altérations génétiques et épigénétiques qui peuvent affecter considérablement le transcriptome. Dans ce sens, le séquençage aléatoire du transcriptome entier des spermatozoïdes ou RNA-seq constitue un outil puissant pour caractériser ces maladies. Jusqu’à présent, il n’existe aucune étude exploitant des données RNA-seq chez des hommes présentant de telles anomalies spermatiques. L’objectif principal de notre étude fût d’identifier des profils distincts des modifications du transcriptome de chaque phénotype d’infertilité pour ainsi révéler des gènes-signatures qui tamponnent une spermatogenèse pathologiquePour ce faire, les transcriptomes des spermatozoïdes de 60 sujets infertiles atteints soit d’oligozoospermie, d’asthénozoospermie ou de tératozoospermie ont été comparés à ceux de 20 patients fertiles. Ces analyses supervisées nous ont conduit à identifier: (i) les gènes clés spécifiques aux différentes anomalies des spermatozoïdes (ii) les voies de signalisation associées, (ii) les différents longs ARNs non codants dérégulés dans ces anomalies. Au niveau de l’oligozoospermie, les transcrits de spermatozoïdes dérégulés étaient associées à divers stades de la spermatogenèse, y compris le cycle cellulaire méiotique, l’assemblage du complexe synaptonémal, la cohésion des chromatides sœurs, les processus métaboliques de piRNA, le processus catabolique protéique dépendant de la voie de l’ubiquitine, à la réponse aux dommages de l'ADN et particulièrement le processus de fécondation. Quant à l’asthenozoospermia, la spermatogenèse, l’assemblage du cil, des voies métaboliques reliées à la spermatogenèse, la chimiotaxie et la physiologie des cellules immunitaires ont été significativement dérégulés. De plus, ce qui nous a intéressé au plus était l’analyse des transcrits sous-exprimés qui a permis l’identification de nombreux transcrits associées aux modifications des histones. Nous avons aussi mis en évidence une sous expression des gènes différentiellement exprimés qui définit la tératozoospermie. Cette sous expression est associée au système ubiquitine-protéasome, à l’organisation du cytosquelette, au cycle cellulaire, à la SUMOylation en réponse aux dommages de l'ADN et aux protéines de réparation ainsi qu’à de nombreux modulateurs épigénétiques. Les gènes signature de l'oligozoospermie ont été liés au processus de fécondation et les composants de la matrice extracellulaire, tandis que ceux de la tératozoospermie sont liés à la spermatogenèse et la morphogenèse cellulaire, alors que les gènes signature de l'asthénozoospermie sont impliqués dans l'assemblage du ribosome et du flagelle. En complément de cette étude, nous avons réalisé une étude très globale du paysage épigénétique du sperme des hommes infertiles. Nous avons, ainsi comparé les niveaux des espèces réactives de l’oxygène (ERO), de méthylation de l’ADN, ainsi que l’intégrité de la chromatine dans les spermatozoïdes de 30 individus infertiles avec ceux de 33 individus fertiles. Nos analyses montrent des niveaux élevés d’ERO chez les individus infertiles. Ces niveaux sont d’une part négativement corrélés avec les niveaux de méthylation globale de l’ADN et d’autre part négativement corrélés avec ceux de la 5-hydroxyméthylcytosine et de la 5-formylcytosine (intermédiaire dans le processus de déméthylation active). Ces derniers suggèrent qu’une infertilité associée au stress oxydatif conditionne l’épigénome du sperme. En conclusion, l’ensemble de notre travail apporte des ressources précieuses et originales dans la compréhension des pathologies de sperme. / Male infertility is actually considered as a public alarming health problem. The sperm pathologies spectrum ranges between different phenotypes including oligozoospermia, asthenozoospermia and teratozoospermia depending on the sperm conventional parameters abnormalities. Abnormal sperm is characterized by genetic alterations and epigenetic alterations which can affect the transcriptome extensively. These alterations in RNA profiles are retrospectively indicative of aberrant spermatogenic events. RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome. To date, RNA-seq analysis of sperm from infertile men has not been reported. Our objectives are: (i) recognize key clusters, key pathways and specific gene transcripts for different sperm abnormalities; (ii) catalog the spermatozoal lncRNAs in different sperm pathologies; (iii) identify signature genes which are mechanistically important in the cascade of events driving a pathological spermatogenesis; (iii) portray the global epigenetic landscape in sperm from infertile men. Expression data from 60 sperm samples from 3 groups of infertile men (oligozoospermia, asthenozoospermia, and teratozoospermia) were generated on Illumina HiSeq platform, compared to 20 fertiles, and the resulting gene expression patterns were analyzed for functional enrichment. Our supervised analyses identified numerous differentially expressed genes between fertile and infertile men. In oligozoospermia, the deregulated spermatozoal transcripts were associated with various stages of spermatogenesis including meiotic cell cycle, synaptonemal complex assembly, sister chromatid cohesion, piRNA metabolic process, ubiquitin-dependent protein catabolic process, cellular response to DNA damage stimulus and interestingly fertilization. As for asthenozoospermia, spermatogenesis, cilium assembly, metabolic-related pathways, chemotaxis and immune cell physiology were most significantly differentially expressed. Interestingly, numerous transcripts associated with histone modifications were highly down-regulated. With regards to teratozoospermia, we evidenced sperm-specific differentially expressed genes which are involved in the ubiquitin-proteasome, cytoskeleton organization, the cell cycle pathway, SUMOylation of DNA damage response and repair proteins, as well as many putative epigenetic modulators of gene expression.. We also attempted to identify distinct patterns of gene expression changes that were definite to the different abnormal sperm phenotypes in infertile men relative to controls. Signature genes of oligozoospermia were over-enriched by genes involved in fertilization and extracellular matrix components, while signature genes of teratozoospermia were enriched by genes involved in spermatogenesis and cellular components involved in morphogenesis, whilst signature genes of asthenozoospermia were enriched by genes implicated in ribosome and cilium assembly.We complemented this work by a parallel epigenetic analysis of the global epigenetic landscape in infertile men. We compared the levels of reactive oxygen species (ROS), DNA integrity and global epigenetic parameters in sperm from 33 infertile subjects with abnormal semen parameters compared to fertile individuals. We pointed out that infertile men are characterized by strikingly high levels of reactive oxygen species (ROS) which were in part negatively correlated with the global DNA methylation, and positively correlated with the levels of 5-hydroxymethylcytosine and 5-formylcytosine (active demethylation intermediates). These findings suggest that male infertility associated with oxidative stress shapes the sperm epigenetic landscape. In summary, this original work yielded a transcriptional portrait of sperm abnormalities and provided valuable resources that would further elucidate sperm pathologies.

One in four known species of fruit flies inhabit the Hawaiian Islands. From a small number of colonizing flies, a wide range of species evolved, some of which managed to reverse-colonize other continental environments. In order to explore the developmental pathways, which separate the Hawaiian Drosophila proper and the Scaptomyza group that contains reverse-colonized species, the transcriptomes of two better-known species in each group, Scaptomyza anomala and Drosophila grimshawi, were analyzed to find changes in gene expression between the two groups. This study describes a novel transcriptome for S. anomala studies as well as unusual changes in gene expression in D. grimshawi relative to other species, revealing priorities of both species in early development.

Sorghum crop has become attractive to breeders due to its drought tolerance, and many uses including a human food source, animal feed, industrial fibre and bioenergy crop. Sorghum, like any other plant, is a host to a variety of microbes that can have neutral, negative or positive effects on the plant. While the majority of microorganisms are beneficial, pathogens colonize plant tissues and overwhelm its defence mechanisms. This colonization is a direct threat to the sorghum productivity. The development of microbiome-based approaches for sustainable crop productivity and yield is hindered by a lack of understanding of the main biotic factors affecting the crop microbiome. Metabarcoding has proven to be a valuable tool which has been widely used for characterizing the microbial diversity and composition of different environments and has been utilized in many research endeavours. This study analyses the relationship between the microbiota and their response to natural pathogen infection in sorghum disease groups (R, MR, S and HS) and identifies the most dominant pathogen in the highly susceptible disease group. The study also, assesses the spore viability through the use of the automated cell counter and confirms Fusarium graminearum (dominant pathogen linked to the HS disease group) through sequencing of the marker genes, to subsequently characterize pathways likely to be involved in pathogen infection resistance. To achieve the objectives, a combination of 16S rRNA (V3/V4 regions) and ITS (ITS1/ITS4) of the internal transcribed spacer regions were amplified and sequenced using NGS technologies to study the microbiota in response to natural infection. Additionally, comparative transcriptional analysis of sorghum RILs in response to Fusarium graminearum infection was conducted through RNA-Seq. Upon natural infection, the foliar symptoms assessment of the RILs was conducted and four disease groups; resistant (R), moderately resistant (MR), susceptible (S) and highly susceptible (HS) were designated. The results of the present metabarcoding study indicate that resistant sorghum leaves (R group) supported a large diversity of fungal and bacterial microbes. The genera Methylorubrum, Enterobacter and Sphingomonas with reported plant growth promoting traits were more abundant and highly enriched in the R and MR group, with members of the latter genus significantly enriched in the R group. The resistant fungal group had a majority of OTUs showing similarity to well-known plant growth-promoting fungal genus including Papiliotrema (Tremellaceae family), which are known biocontrol agents. The yeast Hannaella was also highly linked with the resistant plants. Some Hannaella species are known to produce indole acetic acid (IAA) for promoting plant growth. Metabarcoding was also used to assess the major potential disease-causing taxa associated with the highly diseased group. It identified fungal pathogenic species, that have not previously been identified as pathogens of sorghum such as Ascochyta paspali and Ustilago kamerunensis (which are known pathogenic fungi of grass species) and were associated with the susceptible disease groups (S and HS). These analyses revealed the potential sorghum fungal pathogen Epicoccum sorghinum, and was highly linked with the S disease group. It further expanded the identification of a reportedly economically importance species causing sorghum related diseases Fusarium graminearum (anamorph Gibberella zeae). This species has also been identified in this study to be highly associated with the RILs showing major disease symptoms. Fusarium graminearum a significant pathogen in winter cereals and maize has been associated with stalk rot of sorghum and sorghum grain mould. The presence of Fusarium graminearum in sorghum can be a toxicological risk, since this species has the potential to produce mycotoxins. It was further shown that natural pathogen infection results in distinct foliar microbial communities in sorghum RILs. The co-occurrence taxa represented by Tremellomycetes and Dothiomycetes fungal classes and Bacillaceae and Sphingomonadaceae bacterial family had more central roles in the network. The modules which are located centrally on the network have been expected to play important ‘topological roles’ in interconnecting pairs of other fungal and bacterial taxa in the symbiont–symbiont co-occurrence network. These taxa having a central role, are considered to be keystone microbes, and have been suggested to be drivers of microbiome structure and functioning. The results of bacterial and fungal community composition, community co-occurrences further suggested the importance of keystone taxa which may disproportionately shape the structure of foliar microbiomes. The foliar disease symptom assessments revealed that sorghum RIL 131 was highly diseased and RIL 103 did not show any visible disease symptoms and were subsequently used for transcriptomic analysis. Gene expression patterns were studied between the identified RIL that did not show visible symptoms (resistant RIL no 103) and the RIL that showed major disease symptoms (susceptible RIL no 131). Fusarium graminearum the dominant potential pathogen found in this study to be associated with the highly susceptible plants was used to inoculate RILs at seedling stage in a greenhouse and samples were collected in triplicates at 24 hours post infection (hpi), 48 hpi, 7 days post infection (dpi) and 14 dpi. Prior to that, ITS and UBC genes confirmed the identity of Fusarium graminearum, and the automated haemocytometer confirmed the cell/spore viability. Using RNA-Seq analysis it was shown that the resistant RIL had defence related pathways from early response (24- 48 hpi) to late response (7-14 dpi). And the more the infection progressed, the more the defence related genes were up-regulated in terms of fragments per kilobase of exon model per million reads mapped (FPKM) and False Discovery Rate (FDR ≤ 0.05) values. Transcriptome time series expression profiling was used to characterize the plant response to Fusarium graminearum with the Dirichlet Process Gaussian Process mixture model software (DPGP) in susceptible and resistant RILs. The susceptible RIL (number 131) transcriptional response upon Fusarium graminearum infection presented differences of the closely related clustered expression profiles across all timepoints in both RILs. Group 2 exclusively clustered the genes encoding the sesquiterpene metabolism pathway, which is one of the major physiological change occurring in response to fungal infection and has been previously reported to produce the mycotoxins associated with Fusarium head blight (FHB) of cereals. This pathway presented an increase from the initial infection phase to the late infection phase in group 4, the genes encoding starch sucrose, metabolism and cyanoamino acid pathways presented a pattern that had a sharp decline from 48 hpi -14 dpi (at a later stage of infection). This could suggest that, as the time progresses in the susceptible RIL the pathways which are important in plant defence declines at a late infection stage. Group 3 presented a pattern increase of the 5-lipoxygenase (LOX 5) gene expressed from 48 hpi-14 dpi timepoints. The loss and silencing of LOX5 function have in the past described to be linked with enhanced disease resistance. In this study the LOX5 was expressed and this could suggest that LOX5 might have a function as a susceptibility factor in disease caused by Fusarium graminearum in sorghum RILs. CBL-interacting protein kinase 6 (CIPK6) gene was also associated with this group. This gene has been associated with negative regulation of immune response to Pseudomonas syringae in Arabidopsis as plants overexpressing CIPK6 were more susceptible to Pseudomonas syringae. Transcriptional response of a resistant RIL (number 103) to infection with Fusarium graminearum presented an increase in genes encoding metabolic and biosynthesis of metabolites pathways in group 1 and group 4 at early infection phase and a sharp decline in the late infection phase. An increase in the genes encoding pathways in earlier infection state could suggest the establishment of a beneficial energy balance for defence. Additionally, genes encoding phenylpropanoid (PAL), galactose and glycolysis pathway were amongst the genes increased at early stages of infection in group 1. Sugar can play a significant role in resistance to fungal pathogens through phenylpropanoid metabolism stimulation, and previous studies showed that the phenylpropanoid pathway could play a role in resistance of wheat to Fusarium graminearum and deoxynivalenol. Overall, this study represents a first step in understanding the molecular mechanisms involved in resistance to Fusarium graminearum. This analysis has also identified the reported beneficial microbes and defence related genes and pathways. Together, the current findings suggest that different ‘resident’ consortia found in naturally infected and uninfected sorghum plants may be viable biocontrol and plant-growth promoting targets. Cultivation studies may shed light on the nature of the putative symbiotic relationships between bacteria and fungi. These results have consequences for crop breeding, and the analysis of microbial diversity and community composition can be useful biomarkers for assessing disease status in plants. The transcriptome and metabarcoding data generated will help guide further research to develop novel strategies for management of disease in sorghum RILs through the integrative approach considering both beneficial microbes and defence related genes. This provides the baseline information and will positively impact in the development of Fusarium graminearum resistant genotypes in future through the integration/incorporation of beneficial microorganisms (bacteria and fungi) and resistant genes in breeding strategies. / Life and Consumer Sciences / D. Phil. (Life Sciences)

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