Bien que de nombreuses séquences génomiques soient maintenant connues, les mécanismes évolutifs qui déterminent la taille des génomes, et notamment leur part d’ADN non codant, sont encore débattus. Ainsi, alors que de nombreux mécanismes faisant grandir les génomes (prolifération d’éléments transposables, création de nouveaux gènes par duplication, ...) sont clairement identifiés, les mécanismes limitant la taille des génomes sont moins bien établis. La sélection darwinienne pourrait directement défavoriser les génomes les moins compacts, sous l’hypothèse qu’une grande quantité d’ADN à répliquer limite la vitesse de reproduction de l’organisme. Cette hypothèse étant cependant contredite par plusieurs jeux de données, d’autres mécanismes non sélectifs ont été proposés, comme la dérive génétique et/ou un biais mutationnel rendant les petites délétions d’ADN plus fréquentes que les petites insertions. Dans ce manuscrit, nous montrons à l’aide d’un modèle matriciel de population que la taille du génome peut aussi être limitée par la dynamique spontanée des duplications et des grandes délétions, qui tend à raccourcir les génomes même si les deux types de réarrangements se produisent à la même fréquence. En l’absence de sélection darwinienne, nous prouvons l’existence d’une distribution stationnaire pour la taille du génome même si les duplications sont deux fois plus fréquentes que les délétions. Pour tester si la sélection darwinienne peut contrecarrer cette dynamique spontanée, nous simulons numériquement le modèle en choisissant une fonction de fitness qui favorise directement les génomes contenant le plus de gènes, tout en conservant des duplications deux fois plus fréquentes que les délétions. Dans ce scénario où tout semblait pousser les génomes à grandir infiniment, la taille du génome reste pourtant bornée. Ainsi, notre étude révèle une nouvelle force susceptible de limiter la croissance des génomes. En mettant en évidence des comportements contre-intuitifs dans un modèle pourtant minimaliste, cette étude souligne aussi les limites de la simple « expérience de pensée » pour penser l’évolution. / Even though numerous genome sequences are now available, evolutionary mechanisms that determine genome size, notably their fraction of non-coding DNA, are still debated. In particular, although several mechanisms responsible for genome growth (proliferation of transposable elements, gene duplication and divergence, etc.) were clearly identified, mechanisms limiting the overall genome size remain unclear. Darwinian selection could directly disadvantage less compact genomes, under the hypothesis that a larger quantity of DNA could slow down the speed of reproduction of the organism. Because this hypothesis was proven wrong by several datasets, non selective mechanisms have been proposed, e.g. genetic drift and/or a mutational bias towards small DNA deletions compared to small DNA insertions. In this manuscript, we use a matrix model to show that genome size can also be limited by the spontaneous dynamics of duplications and large deletions, which tends to decrease genome size even if the two types of rearrangements occur at the same rate. In the absence of Darwinian selection, we prove the existence of a stationary distribution of genome size even if duplications are twice as frequent as large deletions. To test whether selection can overcome this spontaneous dynamics, we simulate our model numerically and choose a fitness function that directly favors genomes containing more genes, while keeping duplications twice as frequent as large deletions. In this scenario where, at first sight, everything seems to favor infinite genome growth, genome size remains nonetheless bounded. As a result, our study reveals a new pressure that could be responsible for limiting genome growth. By illustrating counter-intuitive behaviors in a minimal model, this study also underlines the limits of simple "thought experiments" to understand evolution.

Die ALS ist eine fortschreitende neurodegenerative Erkrankung, deren Symptome durch den Untergang der Motoneuronen bedingt sind. Neueste zytogenetische Untersuchungen zeigen ein vermehrtes Auftreten konstitutioneller Chromosomenveränderunge bei ALS-Patienten. Dies lässt eine Verbindung zwischen dem Ausbruch der Erkrankung und der auffälligen Zytogenetik vermuten. Das Auftreten spontaner Chromosomenveränderungen als Zeichen einer chromosomalen Instabilität wurde in der vorliegenden Arbeit an ALS-Patienten untersucht. METHODE: Neben der Karyotypisierung, der Bestimmung der SCE-Rate und der Bruchrate nach Behandlung mit Bleomycin kam die Fluoreszenz in situ Hybridisierung zum Einsatz. Die Untersuchungen wurden an Patienten mit sporadischer ALS (45), an Kontrollpersonen (38) und Verwandten (9) durchgeführt. ERGEBNISSE: Die Karyotypisierung ergab bei den Patienten eine spontane Translokationsrate von 0,02 Translokationen/Zelle (t/Z), bei den Kontrollen 0,04 t/Z. Weitere numerische oder strukturelle Auffälligkeiten waren nicht signifikant verschieden. Es wurden keine konstitutionellen Chromosomenaberrationen gefunden. Die Häufigkeit der Schwesterchromatidaustausche (SCE-Rate) bewegte sich mit 7-8 SCE/Z in der Patientengruppe innerhalb der Normwerte. Durch die Zugabe von Bleomycin in die Zellkultur stieg die Zahl der Chromatidbrüche von 0,0 auf 0,8 Brüche/Z an. Dabei zeigten die untersuchten Gruppen ähnliche Progredienzen in den Bruchraten. Mit der Fluoreszenz in situ Hybridisierung werden quantitative Aussagen über spontane Translokationsraten gemacht. Sie betrug in der Kontroll-und Patientengruppe 0,03 bis 0,04 t/Z. DISKUSSION: Die vorliegenden Ergebnisse liefern keinen Anhalt für eine chromosomale Instabilität als Risikofaktor für die Entstehung der sporadischen ALS. Indizien für eine chromosomale Instabilität wie erhöhte Bruchraten und SCEs als auch vermehrtes Auftreten somatischer Aberrationen konnten bei den ALS-Patienten nicht nachgewiesen werden. Über mögliche Auffälligkeiten in den Motoneuronen lässt sich allerdings mit den Untersuchungen an Blutlymphozyten keine hinreichend sichere Aussage machen. / Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease which is characterized by the degeneration of motor neurons. Recently, a high rate of constitutional structural chromosomal rearrangements has been reported in apparently sporadic ALS patients. It remains questionable whether or not these genomic rearrangements are caused by a chromosomal instability involved in the pathogenesis of the disease. Therefore, we performed different cytogenetic studies on chromosomal instability. METHOD: We performed chromosome analyses from patients (N=45), control subjects (N=38), and relatives (N=9) after culturing blood lymphocytes. Conventional chromosome analysis after GTG-banding, chromosomal breakage test after Bleomycin treatment, the rate of sister chromatid exchange (SCE), and whole chromosome painting were used for these analyses. RESULTS: Neither karyotyping nor whole chromosome painting revealed higher levels of structural or numerical aberrations in lymphocytes of patients with sALS. After karyotyping we found 0.02 t/cell in patients and 0.04 t/cell in controls. Whole chromosome painting revealed 0.04 t/cell in patients and 0.03 t/cell in controls. The chromosomal breaks increased likewise after Bleomycin treatment in the control group and the patient group as well. Cell cultures without Bleomycin did not show any breaks while the highest Bleomycin concentration induced up to 0.08 breaks/cell. The SCE rate in patients which corresponds to the chromatid repair activity did not rise to a higher level than in the control individuals. Both groups were in the normal range of 7 to 8 SCE/cell. DISCUSSION: The pathomechanism of neurodegeneration in ALS patients is still unknown. We tried to find a cytogenetic correlative being a risk factor for the development of ALS. However, so far there is no clue for chromosomal instability being involved in the neurodegenerative process.

Ο καρυότυπος του οικιακού ποντικού Mus musculus domesticus είναι τυπικά ακροκεντρικός εντούτοις χαρακτηρίζεται από τη συχνή εμφάνιση Rb συντήξεων σε φυσικούς πληθυσμούς. Εξαιτίας αυτών μειώνεται ο 2n από τον τυπικό 2n=40 έως και σε 2n=22 και προκύπτουν στη φύση Rb φυλές, οι οποίες όταν έχουν κοινή προέλευση δημιουργούν Rb συστήματα. Στόχος της διδακτορικής διατριβής ήταν ο λεπτομερής καθορισμός της περιοχής εξάπλωσής του Rb συστήματος της Δ. Πελοποννήσου, η Rb σύσταση και οι σχέσεις των Rb φυλών του και η σχέση του με τον ακροκεντρικό πληθυσμό που το περιβάλλει. Επίσης, να προσδιοριστεί η φυλογενετική πορεία που ακολουθήθηκε στο Rb σύστημα και η πιθανή ειδογένεση που δρομολογείται σε αυτό. Γι’ αυτόν το λόγο, έγινε κυτταρολογική μελέτη σε 232 άτομα του ποντικού από 40 τοποθεσίες της Ελλάδας με τη χρήση των τεχνικών G – και C – ζώνωσης. Βρέθηκε ότι το Rb σύστημα της Δ. Πελοποννήσου αποτελείται κυρίως από 3 Rb φυλές με 2n=24, 28 και 30 και έχει ως κέντρο εξέλιξης την περιοχή της Πάτρας, από όπου εκτείνεται για τουλάχιστον 55 km προς τα Β.-ΒΑ. και για πάνω από 70 km προς τα Ν. Οι παραπάνω Rb φυλές συνδέονται φυλογενετικά μέσω 5 κοινών Rb συντήξεων και είναι πιθανό στην εξέλιξη του Rb συστήματος να έχουν συμμετάσχει και Αμοιβαίες Ανταλλαγές Ολόκληρων Βραχιόνων (WART). Ενδέχεται η μετάβαση από το Rb σύστημα στον ακροκεντρικό πληθυσμό να γίνεται απότομα, ενώ ειδογενετικές διαδικασίες θα ήταν δυνατό να πραγματοποιηθούν μεταξύ των Rb φυλών με 2n=24 και 2n=28, που χαρακτηρίζονται από μερική ομολογία βραχιόνων. / The karyotype of the house mouse Mus musculus domesticus is typically acrocentric, yet it is characterized by the frequent appearance of Rb fusions in natural populations. Due to them, 2n is reduced from the typical 2n=40 even down to 2n=22 and Rb races are formed in the nature, which when linked through common decent, form Rb systems. The goal of this doctorate thesis was the detailed definition of the distribution area of the Rb system of W. Peloponnese, of the Rb constitution and relationship among its Rb races and of its relationship with the surrounding acrocentric population. Also, to clarify the phylogenetic process that was followed in this Rb system and the possible speciation that is under way. Thus, a karyological study was implemented on 232 individuals of the house mouse from 40 Greek localities, using the G – and C – banding staining techniques. It was found that the Rb system of W. Peloponnese consists mainly of 3 Rb races with 2n=24, 28 and 30 and its centre of evolution must lie in the wider Patras area, from where it extends for at least 55 km to the N-NE and for more than 70 km to the S. The above Rb races are phylogenetically related through 5 Rb fusions they have in common and it is possible that Whole Arm Reciprocal Translocations (WART) have contributed to the evolution of this Rb system. The transition from the Rb system to the surrounding typical acrocentric population may occur abruptly, whereas speciation processes could take place between the Rb races with 2n=24 and 2n=28, which are charaterized by monobrachial homology.

Made available in DSpace on 2017-07-10T18:13:25Z (GMT). No. of bitstreams: 1 Cassia Fernanda Yano.pdf: 1692770 bytes, checksum: 2f6626f3bb741520d86130373963a00f (MD5) Previous issue date: 2011-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cytogenetics studies were carried out in two genera of fishes from Pindorama and Lopei streams, Upper Paraná River basin, with cytossistematic and ecologic-evolutionary approaches. In Heptapterus mustelinus, the first cytogenetics characterization in the genus was carried out, verifying 54 chromosomes (26m+18sm+4st+6a), which is a diploid number not observed among the Heptapteridae species till now studied. Differently from the others members of this family, multiple Ag-NORs and centromeric heterochromatin in almost all the chromosome of the complement were observed, being these heterochromatin CMA3+/DAPI- in nine chromosome pairs, beyond that coincident with Ag-NORs. These data show a different way in the karyotypic evolution of H. mustelinus in relation to the others Heptapteridae genus. In the genus Astyanax, an interpopulation comparative analysis was carried out in Astyanax aff. paranae, Astyanax fasciatus and Astyanax altiparanae from Pindorama and Lopei streams, in way to observe the chromosomal differentiation and the evolutionary trends in the three species. It was verified 48 and 50 chromosome in Astyanax aff. paranae and A. fasciatus, respectively, with interpopulation differences in the karyotypic formulae. The diploid number obtained to A. altiparanae was 50 chromosomes, with the same karyotypic formulae in both populations. The three species presented multiple Ag-NORs, with interpopulation differences observed in Astyanax aff. paranae and A. fasciatus. In relation to the heterochromatin distribution pattern, interpopulation differences were verified. The occurrence of Ag-NORs/CMA3+ was observed in the three species, besides DAPI+ bands in A. altiparanae. The results showed that Astyanax aff. paranae and A. fasciatus presented more conspicuous interpopulation differences than A. altiparanae populations, probably due bionomic characteristic that avoid the presence of Astyanax aff. paranae and A. fasciatus to upper segments in the streams, leading to the fixation of chromosomal rearrangements in these populations. However, the absence of interpopulation karyotypic differences in A. altiparanae suggests gene flow between the two populations. By the way, the present study showed that cytogenetics is an important tool in understanding the chromosomal mechanism involved in the evolutionary process in fishes group, and that the association to ecological studies is fundamental to the comprehension of dispersion process of some group of fishes. / Estudos citogenéticos foram realizados em dois gêneros de peixes nos riachos Pindorama e Lopei, pertencentes à bacia do Alto rio Paraná, com diferentes abordagens: citossistemática e ecológico-evolutiva. Em Heptapterus mustelinus foi realizada a primeira caracterização citogenética no gênero, tendo sido verificado 54 cromossomos (26m+18sm+4st+6a), número diplóide não relatado entre os heptapterídeos até o momento estudados. Diferentemente da maioria das espécies da família, foram observadas Ag-RONs múltiplas e heterocromatina presente na região centromérica da maioria dos cromossomos do complemento, sendo estas heterocromatinas CMA3+/DAPI- em nove pares cromossômicos, além daquelas coincidentes com as Ag-RONs. Estes dados revelam um caminho diferente na evolução cariotípica de H. mustelinus em relação aos demais gêneros de Heptapteridae. No gênero Astyanax foi realizada uma análise comparativa entre populações de Astyanax aff. paranae, Astyanax fasciatus e Astyanax altiparanae coletadas nos riachos Pindorama e Lopei, verificando-se as tendências da evolução e diferenciação cromossômica nas três espécies. Nas populações de Astyanax aff. paranae e A. fasciatus foram verificados 48 e 50 cromossomos, respectivamente, mas com diferenças interpopulacionais nas fórmulas cariotípicas em ambas as espécies. O número diplóide para A. altiparanae foi de 50 cromossomos com mesma fórmula cariotípica em ambas as populações. As três espécies apresentaram Ag-RONs múltiplas, com diferenças interpopulacionais observadas em Astyanax aff. paranae e A. fasciatus. Em relação ao padrão de distribuição de heterocromatina, diferenças interpopulacionais foram verificadas. A ocorrência de Ag-RONs/CMA3+ foi observada nas três espécies, além de bandas DAPI+ em A. altiparanae. Os dados demonstraram que Astyanax aff. paranae e A. fasciatus apresentaram diferenças cariotípicas interpopulacionais mais conspícuas do que as populações de A. altiparanae, provavelmente por características bionômicas que restringem Astyanax aff. paranae e A. fasciatus a trechos superiores de riachos, com a fixação de rearranjos cromossômicos nas populações. Entretanto, a ausência de diferenças cariotípicas interpopulacionais em A. altiparanae sugerem fluxo entre as duas populações. De forma geral ficou evidente que a citogenética é uma importante ferramenta para o entendimento dos mecanismos cromossômicos envolvidos no processo evolutivo dentro dos grupos de peixes, e que a associação a estudos de ecologia é fundamental para compreender os processos envolvidos na dispersão de algumas espécies.

Such chromosomal rearrangements, which cannot be detected by using of cytogenetic banding of metaphase chromosomes, i.e. chromosomes smaller than 3 - 5 Mb, and therefore modern molecular genetic methods are used to detect them, are called "cryptic rearrangements". Their important role in human pathology is more and more significant. By using of the multiplex ligation-probe dependent amplification method (MLPA) we examined a group of 50 probands with idiopathic mental retardation. A cryptic rearrangement was found at 8 probands (16 %), at 6 of them it was demonstrably causal. Then we examined a group of 40 probands suspected of gene SHOX pathology. A cryptic rearrangement was found at 17 probands (42.5 %) and at 8 of them it was demonstrably causal. Presence of small deletion founded isolated at 7 probands was verified in a population set, but without a positive result. An analysis of mutations was made too.

Les duplicacions segmentàries (DSs), o també anomenades duplicons o Low copy Repeats (LCRs), són regions de coma mínim 1 kb amb un alt nivell d'identitat (>90%), que estan presents almenys dues vegades en el genoma. La regió 8p23.1 consta de 6.5 Mb a la part distal del braç curt del cromosoma 8 i està flanquejada per duplicacions segmentàries. Degut a la seva arquitectura genòmica aquesta regió és susceptible a patir reordenaments mediats per recombinació homòloga no al·lèlica entre les DSs, com per exemple la inversió polimòrfica de 8p23.1 [inv(8)(p23)], present en un de cada quatre individus de la població general europea i japonesa, així com d'altres reorganitzacions menys corrents.El treball realitzat en aquesta tesi doctoral pretén aprofundir en la caracterització de la complexa arquitectura genòmica d'aquesta regió. En la nostra primera aproximació a l'estudi de les DSs que flanquegen la regió cromosòmica 8p23.1, es va identificar una nova família gènica específica de primats, la família gènica FAM90A.Així, bona part d'aquesta tesi doctoral està centrada en l'anàlisi de l'origen, formació, evolució i expansió de FAM90A en els homínids. Per altra banda també s'ha analitzant en detall la variabilitat de FAM90A com a variant en número de còpia (CNV) en diferents poblacions humanes.Finalment, s'ha establert la freqüència de la inversió que afecta a 8p23.1 en població espanyola. També s'ha procedit a genotipar diversos individus homozigots per la inversió i s'ha predit l' estatus de la inversió en 150 individus del projecte HapMap i s'ha analitzat l'efecte que té aquesta reorganització sobre els nivells d'expressió dels gens de la regió. / Segmental duplications (SDs), also known as duplicons or Low Copy Repeats (LCRs), are regions of a minimum of 1 kb with a high sequence identity level (>90%), which are present at least two times in the genome. The 8p23.1 region extends 6.5 Mb at the distal part of the short arm of chromosome 8 and it is flanked by segmental duplications. Due to its genomic architecture the region is prone to suffer rearrangements mediated by non-allelic homologous recombination between these SDs, such as the polymorphic inversion of 8p23.1 [inv(8)(p23)], which is present in one out of every four of European and Japanese general population individuals, as well as other less frequent rearrangements.The aim of the work presented in this doctoral thesis is to get insights in the characterization of the genomic architecture of this complex region. Our first approach to study the SDs flanking 8p23.1 region resulted in the identification of a novel gene family which is primate specific, the FAM90A gene family. Thus, this doctoral thesis is mainly focused on the analysis of the origins, formation, evolution and expansion of FAM90A in hominoids. It has also been analyzed in detail the variability of FAM90A as a copy number variant (CNV) in different human populations.Finally, it has been established the frequency of the inversion affecting 8p23.1 region in the Spanish population. Several homozygous inverted individuals have been genotyped and the status for the inversion has been predicted for 150 HapMap individuals, as well as the effect of this rearrangement on the gene expression levels of the genes contained in the region.

Indiana University-Purdue University Indianapolis (IUPUI) / Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.