Master of Science / Department of Entomology / Jeremy L. Marshall / Sexual conflict is a process that occurs when the evolutionary interests of the sexes is not aligned. The theory began with Darwin in the 1800’s. His observations of sexual dimorphism and traits which did not seem to follow the rules of natural selection led him to the theory of sexual selection. Sexual selection resolved some of the issues which were noted in the evolutionary processes he observed but not all of them. For example, it did not explain traits which increased the male optimum fitness while decreasing the female optimum fitness. It was decades before the concept of sexual conflict was formulated, and even longer before the underlying mechanisms were understood. Sexual conflict is different than sexual selection and there are ways to differentiate which of these processes have occurred in a population. The main forms of sexual conflict are intralocus and interlocus conflict. These involve the interactions between alleles in the genome of the sexes. Intralocus conflict involves conflict at a single locus in the genome while interlocus conflict involves conflict between different loci. Interlocus conflict has been more extensively studied due to its association with sexually antagonistic coevolution (SAC). SAC draws the attention of scientists due to the possibility it is related to the fundamental biological process of speciation. Sexual conflict targets certain traits at defined periods in the mating process. These periods include first, the precopulatory stage which is before the act of copulation begins. Second, there is the stage during copulation but before fertilization of the embryo. Finally, there is the postcopulatory postzygotic stage which is after copulation has ended and fertilization has occurred. Each of these points in the process of mating has traits or behaviors which sexual conflict may target. This review concludes with a proposed experiment to determine if sexual conflict is occurring in a group of four genera of mosquitoes. The experiment utilizes the attributes of sexual conflict to differentiate between other processes. A major component is the consequence of mating systems on selective processes to determine if sexual conflict is involved in the evolution of male accessory gland proteins.

Barley (Hordeum vulgare) is an economically important crop species with a large diploid genome. Around a half of the barley genome and a fifth of the genes are constrained within a low-recombining pericentromeric (LR-PC) region. I explored the LR-PC gene component with a genomic investigation of gene expression, diversity and evolution. Chromatin environments were also explored in the LR and high recombining (HR) regions by surveying the genic and genomic distributions of nine histone modifications. Firstly, regions of HR and LR were identified and compared for gene evolution, expression and diversity. LR regions of the barley genome were found to be restrictive for gene evolution and diversity, but not gene expression. I employed a bioinformatics approach to identify ancient gene pairs in barley to determine the long-term effects of residency in those regions upon gene evolution. Gene pair loss in LR regions was found to be elevated relative to the HR regions. Applying the same method to rice and Brachypodium distachyon revealed the same situation, suggesting a universal process in the grasses for loss of gene pairs in LR regions. The chromosomal distributions of transposable elements (TEs) were also explored and examined for correlations with recombination rate. Secondly, I developed a chromatin immunoprecipitation followed by Next Generation Sequencing (ChIP-seq) protocol for the investigation of histone modifications in barley seedlings. A protocol was optimised for the fixation, extraction and sonication of barley chromatin. The protocol was applied using antibodies against 13 different histone modifications. Following DNA library construction and Illumina sequencing, a bioinformatics pipeline was devised to analyse the sequence data. NGS reads were mapped to a custom assembly of the barley cultivar Morex reference genome sequence before peak calling. Genomic and genic locations were determined for the covalently modified histones. Four modifications were discarded from further study on the basis of low peak numbers or unexpected chromosomal locations. The remaining nine modifications were classified into four groups based on chromosomal distributions. Groupings were closely mirrored by peak sharing relationships between the modifications except histone H3 lysine-27 tri-methylation (H3K27me3). In addition, chromatin states representing local chromatin environments were defined in the barley genome using the peak sharing data. Mapping the states onto the genome revealed a striking chromatin structure of the gene-rich chromosome arms. A telomere-proximal region bearing high levels of H3K27me3-containing states was found adjacent to an interior gene-rich region characterised by active chromatin states lacking H3K27me3. The LTR retroelement-rich interior was found to be associated with repressive chromatin states. The histone modification status of TE classes were also probed revealing unexpected differences relating to the genomic and genic distributions of these elements. Finally, a genome browser was created to host the information publicly.

TOO MANY MOUTHS (TMM) is a gene which has been shown to regulate cell fate and control cell division in the plant leaf epidermis. TMM encodes a leucine rich repeat-receptor like protein (LRR-RLP) which has putative function as a signal transducing factor in the cell to cell signaling pathway involved in controlling proper spacing of stomata! initials throughout the leaf. This gene has been shown to be widely conserved across species of land plants ranging from bryophytes to angiosperms. Curiously, although this gene is highly conserved, stomata! installation pathways vary considerably across these species. Leaf development in both Arabidopsis and monocot grasses has been well characterized, and contrasting stomata! development pathways have been identified between them. Dioscorea bulbifera is a basal member of the monocot family, but this species forms broad leaves similar to many eudicot species. Stomata! development in this species retains some features characteristic of grass installation pathways such as few to no secondary asymmetric divisions of meristemoids. However, new epidermal cell formation occurs by fill-in between leaf veins, more closely resembling broad leaf eudicot development. A TMM ortholog has been isolated in this species and phylogenetic analysis of the sequence may help to place the signals controlling stomata! proliferation in Dioscorea bulbifera into evolutionary perspective.

As sequencing technology has taken off since the late 1990's, a unique phenomenon has been observed repeatedly: genes with little to no conservation across species. For a while, the predominant theory that arose to explain these genes was duplication and subsequent evolution of conserved genes. While this theory can explain some, still many genes have now been proven to arrive de novo - from previously non-coding DNA. This work further investigates the origins of these de novo evolved genes and their practical application relevance in crop biotechnology. This work demonstrates the dynamic nature of plant mitochondrial genomes between even closely related species, and the integral role of mitochondrial genomes in the origin of de novo orphan genes in plants. To better understand the functional potential of plant orphan genes, the network of the orphan gene Qua Quine Starch (QQS) is further elucidated. This analysis demonstrates the broad functionality of an orphan gene as a "fine-tuning knob" in many plant pathways. Further, QQS' role in protein and starch allocation and plant defense is tied to the Nuclear Factor Y subunit C4 (NF-YC4) transcription factor - this knowledge leads us to manipulate expression of native NF-YC4 transcription factors in important crop species to successfully increase seed protein, increase broad disease resistance, and expedite maturation in soybean. A key goal in biotechnology is creating non-transgenic plants with advantageous traits. To accomplish this, the CRISPR-CAS9 system was used to target and delete repressive cis-regulatory elements in the GmNF-YC4-1 promoter sequence. This has allowed us to modify the activity of GmNF-YC4-1 and thus increase soybean seed protein, making it possible to get a non-transgenic plant by segregating out the CRISPR-CAS9 T-DNA and keeping the regulatory deletion. Overall, this work uncovers a novel mechanism of orphan gene evolution and uses the study of the orphan gene QQS to develop important crop biotechnology.

Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.

Elementos transponíveis (TEs) são sequências de DNA que possuem a capacidade de transposição no genoma hospedeiro. O principal objetivo deste trabalho reside na investigação em torno de possíveis contribuições de TEs nos genomas das algas C. reinhardtii e V. carteri, mais especificamente, na arquitetura dos genes ortólogos nestas espécies. Neste contexto, análises em sílico em larga escala foram realizadas, buscando-se identificar associações entre TEs e os genes ortólogos. Os resultados indicaram que os genes em C. reinhardtii tendem a acumular mais cópias de TEs em relação aos seus ortólogos em V. carteri. C. reinhardtii apresentou maior densidade de cópias de TEs para as regiões flanqueadora 5´ , flanqueadora 3´ e intrônica quando comparada a V. carteri; o inverso foi verificado quando analisada a densidade de TEs nas regiões codificantes. Análises para apurar a distribuição dos elementos em regiões intergênicas e intragênicas foram estabelecidas, nas quais a frequência observada dos elementos foi comparada à frequência esperada segundo a distribuição randômica de TEs no genoma, simulada computacionalmente. Foram constatadas regiões em que a presença dos elementos encontra-se significativamente abaixo do esperado, a exemplo de intervalos adjacentes ao início e ao término dos genes, o que provavelmente reflete a seleção negativa de eventos de integração nestas delimitações, em virtude dos efeitos deletérios associados à disrupção de estruturas de regulação da expressão gênica. De forma geral, nas regiões flanqueadoras 5´ e 3´, foi identificada a tendência de elevação da frequência padronizada de TEs à medida que a classe de distância avaliada se distancia do início e do término do gene, respectivamente. A baixa representatividade dos elementos também foi constatada em regiões intragênicas. O estudo da distribuição de TEs nos íntrons dos genes ortólogos indicou a preservação destas regiões quanto à fixação de TEs, sendo a representatividade abaixo do esperado mais evidente em intervalos adjacentes ao éxon, o que minimiza a chance de ruptura no padrão de splicing dos genes. Em sequências codificantes, a escassez de TEs - esperada devido ao provável efeito deletério destes eventos para a função do gene - foi constatada nos ortólogos das duas espécies. No entanto, inovações decorrentes da integração dos elementos em regiões codificantes podem resultar em efeitos evolutivos positivos, embora estes eventos sejam raros. Nas espécies analisadas foram identificados dois casos, de especial interesse, em que um domínio da sequência peptídica encontra-se localizado em região derivada de TE: o primeiro refere-se ao gene Cre06.g262800, em C. reinhardtii, no qual foi identificado o domínio PHD-finger associadao ao elemento Gypsy-5-LTR_CR; o segundo remete ao gene Vocar20001092m.g, em V. carteri, no qual o domínio zinc knuckle foi reconhecido em região derivada do elemento Gypsy3-LTR_VC. Estes genes constituem exemplos da contribuição de TEs na evolução de sequências codificadoras nas espécies C. reinhardtii e V. carteri, corroborando a hipótese de que os TEs podem contribuir na evolução da arquitetura dos genes, apesar do efeito disruptivo inerente à integração dos mesmos em regiões gênicas. / Transposable elements (TEs) are DNA sequences able to transpose in the host genome. The aim of this study resides in the investigation of TEs contributions in the algae C. reinhardtii and V. carteri genomes, more specifically in the architecture of orthologous genes in these species. In this context, large scale in silico analysis were performed to identify associations between TEs and orthologous genes. The results indicated that genes in the C. reinhardtii specie tend to accumulate more TEs copies than orthologous genes in V. carteri. C. reinhardtii showed higher density of TEs copies in the 5´ flanking, 3´ flanking and intronic regions when compared to V. carteri; the opposite was observed in coding regions. Investigation of the elements distribution in the intergenic and intragenic regions was performed, in which the observed TE frequency was compared to expected TE frequency from the simulated random distribution of the elements in the genome. It was verified regions where TE frequency was significantly lower than expected, as in gene boundaries adjacencies, probably reflecting a negative selection of the TE integration events in these delimitations due to deleterious effects associated with disruption of gene regulatory structures. In general terms, it was observed an increasing standardized frequency in the 5´ and 3´ flanking regions as the distance from gene start and gene end, respectively, increases. TEs underrepresentation was also verified in the intragenic regions. The study of TEs distribution in the introns of orthologous genes revealed the preservation of these structures in relation to TEs fixation, with a stronger underrepresentation near exon, which minimizes the chance of gene splicing pattern disruption. In the coding sequences, the TEs scarcity - expected due the likely deleterious effects to gene function - was verified in the orthologous of both species. However, in rare instances, innovations mediated by TEs integration in the coding regions can lead to positive evolutionary effects. In the species analyzed two instances of particular interest were observed, in which the domain of peptide sequence is located in the region derived from TE. The first one refers to the Cre06.g262800 gene, in the C. reinhardtii specie, which has a PHD-finger domain associated with Gypsy-5-LTR_CR element. The second one refers to the Vocar20001092m.g gene, in V. carteri, in which the zinc knuckle was recognized in region derived from Gypsy3-LTR_VC element. These genes are examples of TEs contributions in the evolution of coding sequences in the C. reinhardtii and V. carteri species, corroborating the hypothesis that TEs can contribute to the evolution of gene architecture, despite the inherent disruptive effect in their integration in the gene regions.

Une reconnaissance appropriée des parasites est essentielle pour une réponse immunitaire efficace, assurant l'activation adéquate des mécanismes de défense immunitaire. Chez les vertébrés, il a été démontré que les gènes codant pour les récepteurs de l'immunité adaptative impliqués dans la reconnaissance des agents pathogènes sont soumis à une intense pression sélective. En revanche, beaucoup moins d’études se sont intéressées à la sélection agissant sur les récepteurs de l'immunité innée. Le but de cette thèse est de décrire la variabilité naturelle des gènes de l'immunité innée impliqués dans la détection des agents pathogènes chez les rongeurs et d’analyser les mécanismes responsables de leur évolution. Ce travail s’est focalisé principalement sur les rongeurs de la sous-famille des Murinae et de leur rôle potentiel en tant que réservoirs d’agents pathogènes dangereux pour l’Homme. Tout d´abord nous avons étudié la variabilité intraspécifique de cinq Toll-like récepteurs ciblant les bactéries (TLR1, TLR2, TLR4, TLR5 et TLR6) pour des lignées consanguines de souris domestiques issues d’une population sauvage de deux sous-espèces : Mus musculus domesticus (Mmd) et Mus musculus musculus (Mmm). Les souches consanguines constituent un outil adapté à l'étude de la variabilité des gènes immunitaires car elles confèrent une information sur les allèles présents dans les populations naturelles tout en bénéficiant de génotypes homozygotes. Les résultats les plus significatifs concernent la découverte d'un codon stop dans l'exon 2 du Tlr5 chez une lignée de Mmm et l’absence de variabilité du Tlr4 chez Mmd. A la suite de ces résultats, nous avons décidé de vérifier si l‘absence de polymorphisme du Tlr4 chez Mmd reflète une absence de variabilité dans les populations naturelles, ou si il s’agit plutôt d’un effet de l'échantillonnage ou des croisements ultérieurs. Nous avons donc séquencé le gène Tlr4 pour les deux sous-espèces provenant de la région du Paléarctique Occidentale (au total 39 Mmm et 62 Mmd) puis nous avons comparé ces résultats avec la variabilité génétique d’un gène mitochondrial (cytochrome b). Nous avons confirmé notre prédiction : la variabilité de Tlr4 chez Mmd est fortement réduite par rapport à Mmm, probablement à cause d’agents pathogènes ayant exercé une sélection purifiante chez Mmd durant la colonisation vers l’ouest. Cependant, l'influence de mécanismes évolutifs neutres, tel que la dérive consécutive à un goulot d’étranglement démographique, ne peut être exclue sur la base de nos données. La dernière partie a été consacrée à la comparaison interspécifique de deux récepteurs : TLR4 et TLR7. Ces deux TLRs se différencient à la fois par leur localisation et leur capacité de détection. TLR4 est un TLR extracellulaire reconnaissant principalement les ligands bactériens, essentiellement les lipopolysaccharides, tandis que TLR7 est localisé dans la cellule et détecte les virus à ARN simple brin. L‘objectif était de décrire la variabilité inter-spécifique de chaque récepteur et de révéler les mécanismes de sélection s’exerçant sur ces gènes au cours de leur évolution sur une échelle de temps plus importante. Nous avons analysé 23 espèces de Murinae provenant surtout d’Asie. Nos résultats suggèrent que la sélection purifiante est la force principale ayant agit sur l’évolution des deux TLRs. Cependant, nous avons également mis en évidence des épisodes de sélection diversifiante qui ont pu être à l’origine des variations intra-spécifiques de TLRs observée aujourd’hui chez les rongeurs. Des sites sous sélection positive sont principalement concentrés dans les domaines extracellulaires des deux récepteurs, domaines responsables de la reconnaissance des agents pathogènes. Enfin, la comparaison entre ces deux TLRs montre que le TLR7 est soumis à une sélection négative plus forte. Cette sélection peut s’expliquer en raison des interactions du TLR7 avec les acides nucléiques viraux. / Appropriate recognition of parasites is crucial for effective immune response, ensuring activation of adequate defence mechanisms. In vertebrates, it has frequently been demonstrated that genes encoding proteins involved in pathogen recognition by an adaptive immune system are often subject to intense selection pressures. On the contrary, much less information has been provided on the evolution of recognition mechanisms of innate immunity. The aim of this thesis is to describe the pattern of natural variation of innate immunity genes involved in pathogen recognition in rodents and to analyze the mechanisms of their evolution. We used murine rodents (subfamily Murinae) as a principal model group because they are potential reservoirs of various pathogens dangerous to humans. First, we studied the intraspecific variability of five bacterial sensing Toll-like receptors (TLR1, TLR2, TLR4, TLR5, and TLR6) in inbred strains derived from two subspecies of the house mouse (M. m. musculus, hereafter abbreviated as Mmm and Mus musculus domesticus, Mmd). Wild-derived inbred strains are suitable tools for studying variation of immunity genes because they provide information about alleles that occur in natural populations, and at the same time they occur at homozygous state. The most significant results include the findings of a stop codon in exon 2 of the Tlr5 gene in one Mmm strain and no variability in Tlr4 of Mmd. Following these results we decided to check whether the absence of Tlr4 polymorphism in Mmd reflects the pattern found in natural populations, or whether it is a consequence of insufficient sampling or subsequent breeding. We therefore sequenced Tlr4 in both subspecies across a large part of the Western Palearctic region (in total 39 Mmm and 62 Mmd individuals), then we compared these results with variability on mitochondrial DNA (cytochrome b). The result confirmed our prediction that observed variability in Mmd is strongly reduced also in free-living populations (compared to Mmm), probably due to strong purifying selection by pathogens with which they met during the westward colonization. However, the influence of random evolutionary processes (e.g. drift during bottlenecks) cannot be excluded based on our data. At the intraspecific level, we could not find any sign of positive selection. The last part of my dissertation is devoted to interspecific comparison of two receptors, TLR4 and TLR7. These two TLRs differ in the exposure and the ligands detection. TLR4 is an extracellular receptor detecting mainly bacterial ligands (especially lipopolysaccharides), while TLR7 is located inside the cell and detects ssRNA viruses. The aim of this part of the thesis was to describe variability of both receptors at the interspecific level and to reveal selection forces acting on TLRs in longer evolutionary time scale. In total we analyzed 23 rodent species of the subfamily Murinae in Europe, Asia and Africa. Our results suggest that purifying selection has been a dominant force in evolution of the Tlr4 and Tlr7 genes, but we also demonstrated that episodic diversifying selection has shaped the present species-specific variation in rodent Tlrs. Sites under positive selection were concentrated mainly in the extracellular domain of both receptors, which is responsible for ligand binding. The comparison between two TLRs lead us to the conclusion that the intracellular TLR7 is under much stronger negative selection pressure, presumably due to its interaction with viral nucleic acids.

Elementos transponíveis (TEs) são sequências de DNA que possuem a capacidade de transposição no genoma hospedeiro. O principal objetivo deste trabalho reside na investigação em torno de possíveis contribuições de TEs nos genomas das algas C. reinhardtii e V. carteri, mais especificamente, na arquitetura dos genes ortólogos nestas espécies. Neste contexto, análises em sílico em larga escala foram realizadas, buscando-se identificar associações entre TEs e os genes ortólogos. Os resultados indicaram que os genes em C. reinhardtii tendem a acumular mais cópias de TEs em relação aos seus ortólogos em V. carteri. C. reinhardtii apresentou maior densidade de cópias de TEs para as regiões flanqueadora 5´ , flanqueadora 3´ e intrônica quando comparada a V. carteri; o inverso foi verificado quando analisada a densidade de TEs nas regiões codificantes. Análises para apurar a distribuição dos elementos em regiões intergênicas e intragênicas foram estabelecidas, nas quais a frequência observada dos elementos foi comparada à frequência esperada segundo a distribuição randômica de TEs no genoma, simulada computacionalmente. Foram constatadas regiões em que a presença dos elementos encontra-se significativamente abaixo do esperado, a exemplo de intervalos adjacentes ao início e ao término dos genes, o que provavelmente reflete a seleção negativa de eventos de integração nestas delimitações, em virtude dos efeitos deletérios associados à disrupção de estruturas de regulação da expressão gênica. De forma geral, nas regiões flanqueadoras 5´ e 3´, foi identificada a tendência de elevação da frequência padronizada de TEs à medida que a classe de distância avaliada se distancia do início e do término do gene, respectivamente. A baixa representatividade dos elementos também foi constatada em regiões intragênicas. O estudo da distribuição de TEs nos íntrons dos genes ortólogos indicou a preservação destas regiões quanto à fixação de TEs, sendo a representatividade abaixo do esperado mais evidente em intervalos adjacentes ao éxon, o que minimiza a chance de ruptura no padrão de splicing dos genes. Em sequências codificantes, a escassez de TEs - esperada devido ao provável efeito deletério destes eventos para a função do gene - foi constatada nos ortólogos das duas espécies. No entanto, inovações decorrentes da integração dos elementos em regiões codificantes podem resultar em efeitos evolutivos positivos, embora estes eventos sejam raros. Nas espécies analisadas foram identificados dois casos, de especial interesse, em que um domínio da sequência peptídica encontra-se localizado em região derivada de TE: o primeiro refere-se ao gene Cre06.g262800, em C. reinhardtii, no qual foi identificado o domínio PHD-finger associadao ao elemento Gypsy-5-LTR_CR; o segundo remete ao gene Vocar20001092m.g, em V. carteri, no qual o domínio zinc knuckle foi reconhecido em região derivada do elemento Gypsy3-LTR_VC. Estes genes constituem exemplos da contribuição de TEs na evolução de sequências codificadoras nas espécies C. reinhardtii e V. carteri, corroborando a hipótese de que os TEs podem contribuir na evolução da arquitetura dos genes, apesar do efeito disruptivo inerente à integração dos mesmos em regiões gênicas. / Transposable elements (TEs) are DNA sequences able to transpose in the host genome. The aim of this study resides in the investigation of TEs contributions in the algae C. reinhardtii and V. carteri genomes, more specifically in the architecture of orthologous genes in these species. In this context, large scale in silico analysis were performed to identify associations between TEs and orthologous genes. The results indicated that genes in the C. reinhardtii specie tend to accumulate more TEs copies than orthologous genes in V. carteri. C. reinhardtii showed higher density of TEs copies in the 5´ flanking, 3´ flanking and intronic regions when compared to V. carteri; the opposite was observed in coding regions. Investigation of the elements distribution in the intergenic and intragenic regions was performed, in which the observed TE frequency was compared to expected TE frequency from the simulated random distribution of the elements in the genome. It was verified regions where TE frequency was significantly lower than expected, as in gene boundaries adjacencies, probably reflecting a negative selection of the TE integration events in these delimitations due to deleterious effects associated with disruption of gene regulatory structures. In general terms, it was observed an increasing standardized frequency in the 5´ and 3´ flanking regions as the distance from gene start and gene end, respectively, increases. TEs underrepresentation was also verified in the intragenic regions. The study of TEs distribution in the introns of orthologous genes revealed the preservation of these structures in relation to TEs fixation, with a stronger underrepresentation near exon, which minimizes the chance of gene splicing pattern disruption. In the coding sequences, the TEs scarcity - expected due the likely deleterious effects to gene function - was verified in the orthologous of both species. However, in rare instances, innovations mediated by TEs integration in the coding regions can lead to positive evolutionary effects. In the species analyzed two instances of particular interest were observed, in which the domain of peptide sequence is located in the region derived from TE. The first one refers to the Cre06.g262800 gene, in the C. reinhardtii specie, which has a PHD-finger domain associated with Gypsy-5-LTR_CR element. The second one refers to the Vocar20001092m.g gene, in V. carteri, in which the zinc knuckle was recognized in region derived from Gypsy3-LTR_VC element. These genes are examples of TEs contributions in the evolution of coding sequences in the C. reinhardtii and V. carteri species, corroborating the hypothesis that TEs can contribute to the evolution of gene architecture, despite the inherent disruptive effect in their integration in the gene regions.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24971号 / 医科博第153号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Appropriate recognition of parasites is crucial for effective immune response, ensuring activation of adequate defence mechanisms. In vertebrates, it has frequently been demonstrated that genes encoding proteins involved in pathogen recognition by an adaptive immune system are often subject to intense selection pressures. On the contrary, much less information has been provided on the evolution of recognition mechanisms of innate immunity. The aim of this thesis is to describe the pattern of natural variation of innate immunity genes involved in pathogen recognition in rodents and to analyze the mechanisms of their evolution. We used murine rodents (subfamily Murinae) as a principal model group because they are potential reservoirs of various pathogens dangerous to humans. First, we studied the intraspecific variability of five bacterial sensing Toll-like receptors (TLR1, TLR2, TLR4, TLR5, and TLR6) in inbred strains derived from two subspecies of the house mouse (M. m. musculus, hereafter abbreviated as Mmm and Mus musculus domesticus, Mmd). Wild-derived inbred strains are suitable tools for studying variation of immunity genes because they provide information about alleles that occur in natural populations, and at the same time they occur at homozygous state. The most significant results include the findings of a stop codon in exon 2 of the Tlr5 gene in one Mmm strain and no variability in Tlr4 of Mmd. Following these results we decided to check whether the absence of Tlr4 polymorphism in Mmd reflects the pattern found in natural populations, or whether it is a consequence of insufficient sampling or subsequent breeding. We therefore sequenced Tlr4 in both subspecies across a large part of the Western Palearctic region (in total 39 Mmm and 62 Mmd individuals), then we compared these results with variability on mitochondrial DNA (cytochrome b). The result confirmed our prediction that observed variability in Mmd is strongly reduced also in free-living populations (compared to Mmm), probably due to strong purifying selection by pathogens with which they met during the westward colonization. However, the influence of random evolutionary processes (e.g. drift during bottlenecks) cannot be excluded based on our data. At the intraspecific level, we could not find any sign of positive selection. The last part of my dissertation is devoted to interspecific comparison of two receptors, TLR4 and TLR7. These two TLRs differ in the exposure and the ligands detection. TLR4 is an extracellular receptor detecting mainly bacterial ligands (especially lipopolysaccharides), while TLR7 is located inside the cell and detects ssRNA viruses. The aim of this part of the thesis was to describe variability of both receptors at the interspecific level and to reveal selection forces acting on TLRs in longer evolutionary time scale. In total we analyzed 23 rodent species of the subfamily Murinae in Europe, Asia and Africa. Our results suggest that purifying selection has been a dominant force in evolution of the Tlr4 and Tlr7 genes, but we also demonstrated that episodic diversifying selection has shaped the present species-specific variation in rodent Tlrs. Sites under positive selection were concentrated mainly in the extracellular domain of both receptors, which is responsible for ligand binding. The comparison between two TLRs lead us to the conclusion that the intracellular TLR7 is under much stronger negative selection pressure, presumably due to its interaction with viral nucleic acids.