Recent theoretical developments have led to a renewed interest in the potential role of chromosomal rearrangements in speciation. Australian morabine grasshoppers (genus Vandiemenella, viatica species group) provide an excellent study system to test this potential role, because they show extensive chromosomal variation: 12 chromosomal races/species with parapatric distributions. The research in this thesis involves the application of molecular genetic analyses to examine patterns of gene introgression among chromosomal races of Vandiemenella at three different spatial scales: local-scale hybrid zone analysis, island-scale phylogeography, and continental-scale phylogeography. The aims of these multi-scale analyses are to investigate whether chromosomal races represent genetically distinct taxa with limited gene flow, and to infer the historical biogeography of Vandiemenella and evolutionary origins of their parapatric distributions. Karyotype and 11 nuclear markers revealed a remarkably narrow hybrid zone with substantial linkage disequilibrium and strong deficits of heterozygotes between the chromosome races P24(XY) and viatica17 on Kangaroo Island, suggesting that the zone is maintained by a balance between dispersal and selection against hybrids (tension zone). Selection that maintains the stable hybrid zone is unlikely to be operating only on loci linked to rearranged chromosomes. Island-scale and continental-scale phylogeography using multiple nuclear markers indicated that Vandiemenella chromosome races/species generally represent genetically distinct taxa with reduced gene flow between them. In contrast, analyses of a mitochondrial gene showed the presence of distinctive and geographically localised phylogroups that do not correspond with the distribution of the Vandiemenella taxa. These discordant population genetic patterns are likely to result from introgressive hybridization between the taxa and range expansions and contractions. Overall, our molecular analyses favour the allopatric mode of diversification for the evolution of Vandiemenella and do not support the stasipatric speciation model of White (1978). Patterns of genetic differentiation between the chromosomal races analysed at three different spatial scales show dynamic responses of the grasshoppers to past climatic fluctuations, leading to opportunities for long-term isolation and allopatric fixation of new chromosome variants and molecular mutations at many loci. Further analyses are necessary to assess potential roles of chromosomal rearrangements in facilitating diversification in Vandiemenella by reducing recombination within the rearranged chromosome segments.

O tratamento padrão para pacientes com câncer de reto localmente avançado consiste no uso de quimioradioterapia neoadjuvante (QRTn), seguida por cirurgia. Uma fração significativa dos pacientes responde completamente ao tratamento e no momento da reavaliação não apresenta evidência clínica nem radiológica de doença. Uma abordagem alternativa, Watch and Wait, propõe não operar imediatamente esses pacientes e submetê-los a um protocolo de observação frequente, a fim de evitar as morbidades associadas à cirurgia. No entanto, a avaliação da resposta ao tratamento ainda é um desafio, devido à subjetividade da avaliação clínica e a ausência de exames radiológicos suficientemente sensíveis e específicos para garantir a ausência de células tumorais residuais ou capazes de detectar a recorrência precoce da doença. DNA circulante contendo alterações genéticas específicas do tumor (ctDNA) pode ser encontrado na fração livre de células do sangue e tem sido utilizado para monitorar a dinâmica tumoral em tumores sólidos. Avanços recentes das tecnologias de sequenciamento permitem a identificação eficiente e rápida e a um custo relativamente baixo de alterações genéticas em tumores individuais, superando o problema imposto pela ausência de alterações genéticas recorrentes nesses tumores. Essas alterações podem ser utilizadas como biomarcadores personalizados para monitorar a resposta ao tratamento, detectar doença residual e a recidiva precoce do tumor. O objetivo deste trabalho foi identificar e estudar biomarcadores personalizados em pacientes com câncer de reto localmente avançado tratados com QRTn e avaliar a capacidade desses biomarcadores para monitorar a dinâmica tumoral, e auxiliar na definição da conduta cirúrgica e na detecção da recidiva precoce da doença. Biópsias de seis pacientes com adenocarcinoma de reto distal (cT2- 3N0-1M0), foram coletadas prospectivamente pré-tratamento. O DNA genômico extraído a partir das biópsias foi usado para construir bibliotecas tipo mate-pair para o sequenciamento do genoma completo, utilizando a plataforma SOLiD. Rearranjos inter e intracromossômicos foram identificados utilizando programas computacionais desenvolvidos pelo nosso grupo de pesquisa e em seguida foram validados utilizando PCR e sequenciamento Sanger. Foram validadas, pelo menos, três variações estruturais para cada paciente. Amostras de plasma foram coletadas no momento do diagnóstico, depois da QRTn e durante o seguimento. DNA circulante total foi extraído a partir das amostras de plasma e ensaios personalizados foram desenvolvidos para monitorar a presença de variações estruturais através de PCR Digital. ctDNA foi detectado em todas amostras de plasma pré-tratamento de pacientes com tumores T3. A detecção desses biomarcadores apresentou boa correlação com a resposta ao tratamento, no entanto, esta abordagem não foi sensível o suficiente para detectar doença residual. Para dois pacientes que desenvolveram doença metastática foi verificado um aumento nos níveis de ctDNA com pelo menos 36 semanas antes do diagnóstico clínico de doença metastática, sendo possível correlacionar os níveis de ctDNA detectados em coletas subsequentes com a resposta ao tratamento sistêmico de segunda linha. Este estudo, embora de caráter exploratório, gerou dados relevantes e suficientes para justificar a realização de estudos adicionais para avaliar a aplicação dos biomarcadores personalizados na definição da conduta cirúrgica e no acompanhamento de pacientes com câncer de reto tratados com QRTn. / The standard treatment for patients with locally advanced rectal cancer comprises in neoadjuvant chemo radiotherapy (nCRT), followed by surgery. A significant fraction of these patients show complete response to the treatment and at the time of reassessment, there are no clinical and nor radiological evidence of residual tumor. An alternative approach, Watch and Wait, proposes not to immediately operate these patients, but to submit them to a protocol of frequent observation in order to avoid the morbidities associated with radical surgery. However, assessment of treatment response remains a significant challenge due to the subjectivity of the clinical examination and to the lack of sufficiently sensitive tools to ensure the absence of tumor cells or to detect early disease recurrence. Circulating DNA carrying tumor-specific genetic alterations (circulating tumor DNA - ctDNA) can be found in the cell-free fraction of the blood and has been successfully used to monitor the tumor dynamics in solid tumors. Recent advances in sequencing technologies have enabled the rapid and cost effective identification of genetic alterations in individual tumors, overcoming the problem imposed by the absence of recurrent genetic alterations in these tumors. These alterations can be used as personalized biomarkers to monitor treatment response, detect residual disease and early tumor recurrence. The purpose of this work was to identify and validate the use of personalized biomarkers for patients with locally advanced rectal cancer treated with nCRT and to evaluate the ability of these biomarkers to monitor the tumor dynamics, to define surgical approach and to detect early recurrence of the disease. Pre-treatment biopsies from 6 patients with cT2-3N0-1M0 distal rectal adenocarcinoma were prospectively collected. Genomic DNA extracted from the biopsies was used to construct mate-pair libraries for whole genome sequencing using SOLiD platform. Inter and intrachromosomal rearrangements were identified using an in-house bioinformatics pipeline and validated using PCR amplification and Sanger sequencing. At least three structural variations were validated for each patient. Plasma samples were collect at diagnosis, after nCRT and follow-up. Circulating DNA was obtained from the plasma samples and personalized assays were designed to monitor the presence of structural variations using Droplet Digital PCR. ctDNA was detected in all pre-treatment plasma samples for patients with T3 tumors. The detection of these biomarkers showed a good correlation with the treatment response, nonetheless, the approach was not sensitive enough to detect residual disease. In two patients who developed metastatic disease, an increase in ctDNA levels was observed at least 36 weeks before clinical detection of metastatic disease, and it was possible to correlate the level of ctDNA in subsequent plasma samples with response to the second-line treatment. This study, although exploratory, generated relevant and sufficient data to support additional studies to evaluate the use of personalized biomarkers in the surgical management and follow-up of rectal cancer patients treated with nCRT.

O tratamento padrão para pacientes com câncer de reto localmente avançado consiste no uso de quimioradioterapia neoadjuvante (QRTn), seguida por cirurgia. Uma fração significativa dos pacientes responde completamente ao tratamento e no momento da reavaliação não apresenta evidência clínica nem radiológica de doença. Uma abordagem alternativa, Watch and Wait, propõe não operar imediatamente esses pacientes e submetê-los a um protocolo de observação frequente, a fim de evitar as morbidades associadas à cirurgia. No entanto, a avaliação da resposta ao tratamento ainda é um desafio, devido à subjetividade da avaliação clínica e a ausência de exames radiológicos suficientemente sensíveis e específicos para garantir a ausência de células tumorais residuais ou capazes de detectar a recorrência precoce da doença. DNA circulante contendo alterações genéticas específicas do tumor (ctDNA) pode ser encontrado na fração livre de células do sangue e tem sido utilizado para monitorar a dinâmica tumoral em tumores sólidos. Avanços recentes das tecnologias de sequenciamento permitem a identificação eficiente e rápida e a um custo relativamente baixo de alterações genéticas em tumores individuais, superando o problema imposto pela ausência de alterações genéticas recorrentes nesses tumores. Essas alterações podem ser utilizadas como biomarcadores personalizados para monitorar a resposta ao tratamento, detectar doença residual e a recidiva precoce do tumor. O objetivo deste trabalho foi identificar e estudar biomarcadores personalizados em pacientes com câncer de reto localmente avançado tratados com QRTn e avaliar a capacidade desses biomarcadores para monitorar a dinâmica tumoral, e auxiliar na definição da conduta cirúrgica e na detecção da recidiva precoce da doença. Biópsias de seis pacientes com adenocarcinoma de reto distal (cT2- 3N0-1M0), foram coletadas prospectivamente pré-tratamento. O DNA genômico extraído a partir das biópsias foi usado para construir bibliotecas tipo mate-pair para o sequenciamento do genoma completo, utilizando a plataforma SOLiD. Rearranjos inter e intracromossômicos foram identificados utilizando programas computacionais desenvolvidos pelo nosso grupo de pesquisa e em seguida foram validados utilizando PCR e sequenciamento Sanger. Foram validadas, pelo menos, três variações estruturais para cada paciente. Amostras de plasma foram coletadas no momento do diagnóstico, depois da QRTn e durante o seguimento. DNA circulante total foi extraído a partir das amostras de plasma e ensaios personalizados foram desenvolvidos para monitorar a presença de variações estruturais através de PCR Digital. ctDNA foi detectado em todas amostras de plasma pré-tratamento de pacientes com tumores T3. A detecção desses biomarcadores apresentou boa correlação com a resposta ao tratamento, no entanto, esta abordagem não foi sensível o suficiente para detectar doença residual. Para dois pacientes que desenvolveram doença metastática foi verificado um aumento nos níveis de ctDNA com pelo menos 36 semanas antes do diagnóstico clínico de doença metastática, sendo possível correlacionar os níveis de ctDNA detectados em coletas subsequentes com a resposta ao tratamento sistêmico de segunda linha. Este estudo, embora de caráter exploratório, gerou dados relevantes e suficientes para justificar a realização de estudos adicionais para avaliar a aplicação dos biomarcadores personalizados na definição da conduta cirúrgica e no acompanhamento de pacientes com câncer de reto tratados com QRTn. / The standard treatment for patients with locally advanced rectal cancer comprises in neoadjuvant chemo radiotherapy (nCRT), followed by surgery. A significant fraction of these patients show complete response to the treatment and at the time of reassessment, there are no clinical and nor radiological evidence of residual tumor. An alternative approach, Watch and Wait, proposes not to immediately operate these patients, but to submit them to a protocol of frequent observation in order to avoid the morbidities associated with radical surgery. However, assessment of treatment response remains a significant challenge due to the subjectivity of the clinical examination and to the lack of sufficiently sensitive tools to ensure the absence of tumor cells or to detect early disease recurrence. Circulating DNA carrying tumor-specific genetic alterations (circulating tumor DNA - ctDNA) can be found in the cell-free fraction of the blood and has been successfully used to monitor the tumor dynamics in solid tumors. Recent advances in sequencing technologies have enabled the rapid and cost effective identification of genetic alterations in individual tumors, overcoming the problem imposed by the absence of recurrent genetic alterations in these tumors. These alterations can be used as personalized biomarkers to monitor treatment response, detect residual disease and early tumor recurrence. The purpose of this work was to identify and validate the use of personalized biomarkers for patients with locally advanced rectal cancer treated with nCRT and to evaluate the ability of these biomarkers to monitor the tumor dynamics, to define surgical approach and to detect early recurrence of the disease. Pre-treatment biopsies from 6 patients with cT2-3N0-1M0 distal rectal adenocarcinoma were prospectively collected. Genomic DNA extracted from the biopsies was used to construct mate-pair libraries for whole genome sequencing using SOLiD platform. Inter and intrachromosomal rearrangements were identified using an in-house bioinformatics pipeline and validated using PCR amplification and Sanger sequencing. At least three structural variations were validated for each patient. Plasma samples were collect at diagnosis, after nCRT and follow-up. Circulating DNA was obtained from the plasma samples and personalized assays were designed to monitor the presence of structural variations using Droplet Digital PCR. ctDNA was detected in all pre-treatment plasma samples for patients with T3 tumors. The detection of these biomarkers showed a good correlation with the treatment response, nonetheless, the approach was not sensitive enough to detect residual disease. In two patients who developed metastatic disease, an increase in ctDNA levels was observed at least 36 weeks before clinical detection of metastatic disease, and it was possible to correlate the level of ctDNA in subsequent plasma samples with response to the second-line treatment. This study, although exploratory, generated relevant and sufficient data to support additional studies to evaluate the use of personalized biomarkers in the surgical management and follow-up of rectal cancer patients treated with nCRT.

Made available in DSpace on 2017-07-21T19:59:47Z (GMT). No. of bitstreams: 1 Larissa Glugoski.pdf: 3355270 bytes, checksum: c1790717a7cb103b4caf05eb10cb56cb (MD5) Previous issue date: 2017-02-23 / The Loricariidae family is the largest in the Siluriformes order, being comprised of eight subfamilies. One of these, the Loricariinae subfamily, shows great diversity in respect to the number of chromosomes and karyotype formula, varying in the diploid number (2n) from 36 to 74 chromosomes. This diverse range originated mainly from Robertsonian(Rb) rearrangements. Rineloricaria is the largest genre in the Loricariinae subfamily, its species ranging from 2n = 36 to 70 chromosomes. In spite of this, little is known about which kinds of repetitive DNA gave rise to the events of chromosome fusion or fission. Previous studies have revealed the presence of multiple 5S rDNA sites in specimens of Rineloricaria from the Paraná River Basin, associated to the Robertsonian fission/fusion events. The aim of this work was the molecular characterization of the fragile sites associated to the 5S rDNA, besides localizing in situ marker chromosomes in Rineloricaria latirostris from the Das Pedras River and R. latirostris from the Piumhi River (first described in this work), seeking to understand the 2n diversification in this group. Rineloricaria latirostris from the Pedras River exhibited 2n = 46 chromosomes, while those from the Piumhi River presented 2n = 48 chromosomes, and both had a fundamental number (FN) of 60. Fluorescence in situ hybridization (FISH) assays in R. latirostris from the Piumhi River revealed 2 chromosome pairs with 5S rDNA sites, pair 7 with 18S rDNA, and only terminal staining when subjected to a telomeric probe (TTAGGGn). The population of the Pedras river exhibited 5 pairs with 5S rDNA sites, the metacentric (m) pair 2 marked with 18S rDNA, TTAGGGn markers in the terminal regions of the chromosomes, and the presence of interstitial telomeric sites (ITS) in pairs m 1 and m 3. The latter, in synteny with 5S rDNA, is indicative of Robertsonian fusion events. The isolation, cloning and sequencing of the 5S rDNA revealed clones with high sequence identity to 5S rDNA from other species, in addition to the necessary regions for recognition and transcription by RNA polymerase III. One clone of ~700 bp exhibited a degenerated fragment of hAT transposon in its sequence. It was named degenerated 5S rDNA. The fluorescence in situ hybridization assay highlighted chromosomes with co-localized staining for 5S rDNA/hAT, 5S rDNA/degenerated 5S rDNA, and 5S rDNA/ITS (m 3 pair) in R. latirostris from das Pedras River. In R. latirostris from Piumhi River, there was no detection of degenerated 5S rDNA sites. These results allow us to infer the role of the hAT transposon in the dispersion of 5S rDNA sites in the population, since some studies have indicated a relation between 5S rDNA dispersion and transposons in fish. In conclusion, data obtained by this study indicate a possible association between the hAT and the dispersion of 5S rDNA sites and Robertsonian events in the studied population of R. latirostris. The presence of the 5S rDNA/degenerated 5S rDNA/ITS generates hotspots for chromosomal breakage, contributing to the large karyotype diversity found in Loricariidae. / A família Loricariidae é a mais numerosa dentro da ordem Siluriformes e abrange oito subfamílias. A subfamília Loricarinae apresenta uma grande diversidade no que diz respeito ao número de cromossomos e a fórmula cariotípica, com variação do número diploide (2n) de 36 a 74 cromossomos, sendo os rearranjos Robertsonianos (Rb) considerados os principais mecanismos para explicar esta variação cromossômica. Rineloricaria é o gênero mais numeroso de Loricariinae, com espécies apresentando 2n = 36 - 70 cromossomos. Contudo, pouco ainda se sabe sobre quais os tipos de DNAs repetitivos originaram os eventos de fissão e fusão cromossômica. Estudos anteriores revelaram a presença de sítios múltiplos de rDNA 5S em exemplares de Rineloricaria da bacia do Rio Paraná, associados aos eventos de fissão/fusão Robertsonianos. O objetivo deste trabalho foi a caracterização molecular de sítios frágeis associados ao rDNA 5S, além da localização in situ de marcadores cromossômicos em Rineloricaria latirostris do rio das Pedras e R. latirostris do rio Piumhi (pela primeira vez descrito neste trabalho), visando a compreensão da diversificação do 2n neste grupo. Rineloricaria latirostris do rio das Pedras apresentou 2n = 46 cromossomos, enquanto R. latirostris do rio Piumhi apresentou 2n = 48 cromossomos, ambos com número fundamental (NF) de 60. Ensaios de hibridação in situ fluorescente em R. latirostris do rio Piumhi revelaram 2 pares cromossômicos marcados com rDNA 5S, o par 7 marcado com rDNA 18S, além de apenas marcações terminais utilizando-se a sonda telomérica (TTAGGGn). A população do rio das Pedras apresentou 5 pares portadores de sítios de rDNA 5S, o par metacêntrico (m) 2 marcado com rDNA 18S, marcações de TTAGGGn nas regiões terminais dos cromossomos, além da presença de vestígios de sítios teloméricos intersticiais (interstitial telomeric sites - ITS) nos pares m 1 e m 3, sendo este último em sintenia com o rDNA 5S, indicativo de eventos de fusão Robertsoniana. O isolamento, clonagem e sequenciamento de fragmentos de rDNA 5S, revelaram clones apresentando alta identidade ao rDNA 5S de outras espécies, além das regiões necessárias para o reconhecimento e transcrição pela RNA polimerase III. Um dos clones de ~700 pb apresentou um fragmento do transposon hAT em sua sequência, já em intensa degeneração molecular, sendo denominado de rDNA 5S degenerado. A hibridação in situ fluorescente evidenciou cromossomos com marcações co-localizadas de rDNA 5S/hAT, rDNA 5S/rDNA 5S degenerado e rDNA 5S/ITS (no par m 3) em R. latirostris do rio da Pedras. Em R. latirostris do rio Piumhi, não foram detectados sítios com rDNA 5S degenerado. Estes resultados nos permitem inferir o papel do TE hAT na dispersão dos sítios de rDNA 5S na população estudada, visto que alguns estudos indicam haver uma relação entre a dispersão do rDNA 5S pelo genoma e TEs em peixes. Em conclusão, os dados obtidos neste estudo indicam uma possível associação entre o elemento hAT e a dispersão de sítios de rDNA 5S e eventos Robertsonianos presentes na população de R. latirostris estudada. A presença de rDNA 5S/rDNA 5S degenerado/ITS geram hotspots para as quebras cromossômicas, contribuindo assim para a ampla diversidade cariotípica encontrada em Loricariidae.

Le gène ALK code pour un récepteur à activité tyrosine kinase exprimé principalement dans le système nerveux central et périphérique au cours du développement chez le mammifère. Ces informations font suspecter un rôle développemental du récepteur ALK bien que ses fonctions précises ne sont pas connues. De la même manière, ses ligands et les voies de signalisation qui lui sont associés demeurent mal caractérisés. En 2008, des mutations ponctuelles du gène ALK ont été identifiées dans des formes sporadiques et familiales de neuroblastome. Deux hotspots de mutation sont situés dans le domaine tyrosine kinase. Ces travaux de thèse ont permis d’étudier l’implication du gène ALK dans l’oncogenèse du neuroblastome sous des aspects cellulaires, génomiques et fonctionnels. L’étude de la régulation de l’activation et de l’adressage de la protéine ALK a révélé qu’à la différence des récepteurs sauvages, l’activation constitutive conduit à une rétention intracellulaire associée à une maturation incomplète des récepteurs mutés. Par ailleurs, la caractérisation complète du locus ALK dans le neuroblastome a mis en évidence une fréquence élevée de réarrangements chromosomiques. Ce travail a notamment identifié un réarrangement chromosomique conduisant à l’expression d’un variant du récepteur ALK tronqué pour une partie du domaine extracellulaire. Ce variant, associé à l’agressivité tumorale, est activé de façon constitutive et présente des propriétés oncogéniques. Enfin, l’analyse de souris knock-in exprimant les mutations AlkF1178L et AlkR1279Q a révélé un rôle majeur du gène Alk dans le développement du système nerveux sympathique. En effet, ces souris présentent une hyperplasie des ganglions sympathiques mise en place chez l’embryon et associée à une augmentation de la prolifération cellulaire à la naissance. Ce travail a aussi mis en évidence la coopération oncogénique in vivo entre les mutations du gène Alk et la surexpression du gène MYCN dans le neuroblastome. Les tumeurs générées chez les souris partageant ces deux altérations se développent après une courte période de latence et avec une pénétrance complète. L’analyse des tumeurs a mis en évidence des propriétés conférées aux tumeurs par les mutations du gène Alk. En effet, ces tumeurs expriment des marqueurs cholinergiques et montrent des signes de différenciation. Ces animaux permettent de mieux comprendre le rôle du gène ALK dans l’oncogenèse du neuroblastome et constituent de bons modèles d’étude préclinique des neuroblastomes dépendants de ALK. / The ALK protein is a receptor tyrosine kinase mainly expressed in the central and peripheral nervous system during the development in mammals. These informations suggest that the ALK receptor might have a developmental role even though its functions remain unknown. Ligands and signaling pathways associated to this receptor are also poorly characterized. In 2008, point mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma with two main hotspots of mutation in the kinase domain. This thesis project allowed to study the involvement of the ALK gene in neuroblastoma oncogenesis under cellular, genomic and functional aspects.The study of ALK protein activation and subcellular localization revealed that the constitutive activation of the kinase domain lead to an intracellular retention and to a lack of glycosylation. The genomic characterization of the ALK locus pointed out the high frequency of chromosomal rearrangements in neuroblastoma cell lines. This work notably identified the expression of a chromosomal rearrangement leading to a truncated protein variant of ALK lacking a part of the extracellular domain. This variant, associated with the tumoral aggressiveness, is constitutively activated and harbor oncogenic properties. Finally, the analysis of the knock-in mice expressing the two AlkF1178L and AlkR1279Q mutations demonstrated that the ALK gene has a key role in the regulation of the developing sympathetic nervous system. Indeed, these mice have an hyperplasia of sympathetic ganglia set up during embryogenesis and associated with an increased neuroblast proliferation at birth. This work also described the oncogenic cooperation between ALK mutations and MYCN overexpression in neuroblastoma. Tumors, generated in mice sharing those two alterations, arise with a full penetrance and a short latency. Histological and transcriptomic analysis of tumors identified specific properties provided by ALK mutations. Indeed, these tumors express cholinergic markers and harbor signs of differentiation. Those animals allow to better understand the role of the ALK gene in neuroblastoma oncogenesis and constitute good preclinical models for ALK dependant neuroblastoma.

The process of evolution is of both scientific and medical interest. This thesis presents several studies using complete genomic reference sequences, comparative genomic data, and intraspecific diversity data to study the two key processes of evolution: mutation and selection. Large duplications, deletions, inversions, and translocations of DNA contribute to genomic variation both between and within species. Human chromosomes 15 and 17 contain a high percentage of dispersed, recently duplicated sequences. Examination of the relationships between these sequences showed that the majority of all duplications within each chromosome could be linked through core sequences that are prone to duplication. Comparison to orthologous sequences in other mammals allowed a reconstruction of the ancestral state of the human chromosomes, revealing that regions of rearrangement specific to the human lineage are highly enriched in chromosome-specific duplications. Comparison to copy number variation data from other studies also shows that these regions are enriched in current human structural variation. One specific region, the MAPT locus at 17q21.31, known to contain an inversion polymorphism in Europeans, was resequenced completely across both human orientation haplotypes and in chimpanzee and orangutan, revealing complex duplication structures at the inversion breakpoints, with the human region being more complex than chimpanzee or orangutan. Fluorescent in-situ hybridization analysis of human, chimpanzee, and orangutan chromosomes showed inversion polymorphisms of independent origin in all three species, demonstrating that this region has been a hotspot of genomic rearrangement for at least twelve million years. These results reveal a mechanistic relationship between sequence duplication and rearrangement in the great apes. We also generated a draft sequence of the chimpanzee genome and compared it to that of the human. Among other findings, this showed that CpG dinucleotides contribute 25% of all single base mutations, with a rate of mutation ~10-fold that of other bases, and that the male mutation rate in great apes is ~5-6 times the female rate, a higher ratio than had been observed in comparisons of primates and rodents. We detected six regions of probable recent positive selection in humans with a statistical method relying on chimpanzee sequence to control for regional variation in mutation rates. Finally, resequencing of several lines of domestic chicken and comparison to the reference chicken genome identified a number of gene deletions fixed in domestic lines and also several potential selective sweeps. Of particular interest are a missense mutation in TSHR nearly fixed in all domestic chickens and a partial deletion of SH3RF2 fixed in a high growth line. The TSHR mutation may play a role in relaxation of seasonal reproduction. A high-resolution QTL mapping experiment showed that the SH3RF2 deletion is significantly associated with increased growth. This work provides important new insights into the mechanics of evolutionary change at both the single nucleotide and structural level and identifies potential targets of natural and artificial selection in humans and chickens.

The Araneoidea superfamily is a diverse clade of spiders with a great species diversity. The whole superfamily displays considerable conservativeness of observed karyotypes. Most likely ancestral karyotype in males is 24 acrocentric chromosomes with X1X2 sex determination system. The goal of this study is to explore the karyotype diversity of two araneoid families - Araneidae and Mimetidae. The majority of studied species exhibit the ancestral karyotype. In some species of the aformentioned families was observed sudden increase in chromosome numbers, up to 2n♂ = 52 in Araneidae and up to 2n♂ = 57 in Mimetidae. The latter number is the highest chromosome count observed in Entelegynae so far. Increase in 2n goes hand in hand with increase in sex chromosome numbers, leading up to X1X2X3X40 system in Araneidae and up to X1X2X3X4X5X6X70 in Mimetidae. I suggest polyploidy as a possible mechanism of the increase. To test this hypothesis, I measured the size of the genome using flow cytometry and used fluorescence in situ hybridization for the detection of 18S rRNA and 5S rRNA genes. For one species, probe for U2 snRNA gene was also optimized as part of this thesis. In many species studied, these techniques were used for the first time ever. In the case of the family Mimetidae, the largest genomes in...

Les réarrangements permettent une évolution rapide du génome par l’acquisition de séquences codantes exogènes, la perte de fonctions non-essentielles ou la création de nouvelles organisations génomiques. Différents mécanismes de réarrangements impliquant des éléments génétiques mobiles (EGM) ont été identifiés chez les archées, les bactéries et les eucaryotes. En revanche, on ignore l’origine des nombreuses inversions génomiques détectées pour les espèces du genre archéen Thermococcus. Mes travaux de thèse visent à améliorer la compréhension de l’évolution génomique chez les Thermococcales à travers l’étude de deux familles d’EGM : les familles de plasmides pTN3 et pT26-2. Plus précisément, je me suis intéressée aux recombinases à tyrosine (ou intégrases) que ces plasmides encodent et qui permettent leur intégration dans le chromosome de l’hôte. J’ai montré que l’intégrase plasmidique Intᵖᵀᴺ³ est responsable d’inversions dans le chromosome de son hôte Thermococcus nautili grâce à une activité catalytique inédite de recombinaison homologue. J’ai par la suite caractérisé deux autres intégrases de Thermococcales reliés phylogénétiquement à Intᵖᵀᴺ³ dont seulement une présente une activité de recombinaison homologue. La comparaison de leurs séquences primaires et la résolution de la structure de Intᵖᵀᴺ³ vont maintenant éclairer les déterminants génétiques responsables de la spécificité de site et de l’activité de recombinaison homologue. Les trois intégrases appartiennent à une classe de recombinases spécifique des archées qui catalyse une intégration suicidaire. Lors de l’intégration, le gène de l’intégrase est fragmenté et probablement désactivé. L’EGM intégré se retrouve piégé dans le chromosome. Les avantages évolutifs d’une telle activité suicidaire restent pour l’instant mystérieux. J’ai identifié 62 intégrases hyperthermophiles suicidaires et reconstruit leur histoire évolutive. Ces intégrases sont très prévalentes et recrutées par différents EGM. De plus, j’ai montré que l’une de ces intégrases présente in vitro une activité de recombinaison site-spécifique à des températures proches de l’ébullition de l’eau, représentant un avantage dans les environnements hyperthermophiles. / Genomes rapidly evolve through rearrangements that can generate new genome organizations or lead to the acquisition of foreign coding sequences or the loss of non-essential functions. Several mechanisms of rearrangement were uncovered for Archaea, Bacteria and Eukaryotes that involve mobile genetic elements (MGE). Species from the archaeal genera Thermococcus present numerous genomic inversions but none of the previously known inversion drivers. To better understand the genomic evolution of Thermococcales, I investigated two of their MGE families: the pTN3 and pT26-2 plasmid families. Specifically, I focused on the tyrosine recombinases (or integrase) that these plasmids encode and that catalyze their site-specific integration in the host chromosome. I demonstrated that the plasmidic integrase Intᵖᵀᴺ³ is responsible for chromosomal inversions in the host Thermococcus nautili through an unprecedented homologous recombination catalytic activity. I also characterized two other related Thermococcus integrases and only one catalyzes homologous recombination. The structure resolution of Intᵖᵀᴺ³ and primary sequence comparisons will now provide clues about the genetic determinants of site specificity and of the homologous recombination activity. The three integrases all belong to an archaeal-specific class of integrases that catalyzes a suicidal integration. The integrase gene is partitioned and presumably inactivated upon integration. The integrated MGE is then trapped into the chromosome. The evolutionary benefits of this suicide activity are puzzling. I identified 62 related suicidal hyperthermophilic integrases and reconstructed their evolutionary history. They are highly prevalent and recruited by diverse MGE. I also showed that one of these integrases can catalyze in vitro site-specific recombination at near boiling water temperature, representing an advantage in hyperthermophilic environments.