Student Number : 9901196J - MSc dissertation - School of Pathology - Faculty of Science / Fragile sites are highly unstable regions of the genome, which have a tendency to form gaps and breaks in metaphase chromosomes under replication stress conditions. There are many common fragile sites in the human genome and exposure to carcinogens may affect several genes localised in fragile sites within a single cell, which could lead to activation of oncogenes and inactivation of tumour-suppressor genes simultaneously. FRA3B on chromosome 3 and FRA16D on chromosome 16 are the two most commonly expressed fragile sites and contain the FHIT and WWOX genes respectively. These genes are tumour suppressor genes and are inactivated in a number of different ways. Carcinogens found in cigarette smoke have been found to increase fragile site expression and could alter the integrity of theses genes in active smokers. Ten healthy non-smoking (control) individuals and twenty active smokers were recruited for the purpose of this study. Fluorescence in situ hybridisation was performed with probes spanning spanning the FHIT gene and RT-PCR was performed to assess both FHIT and WWOX expression. No significant difference in breaks at fragile sites was observed between controls and active smokers in the FISH experiments. In addition, no aberrant transcripts were detected for either FHIT or WWOX with RT-PCR. Although the sampling group was limited and heterogenous, no increase in the expression of breaks at fragile sites was seen in active smokers in the present study.

WWOX (WW domain-containing oxidoreductase) is a candidate tumor suppressor gene that has been shown to be involved in various cancers including breast, lung, prostate, gastric and hepatic. The Drosophila ortholog Wwox was identified and subjected to targeted ‘loss of function’ mutagenesis. The resulting mutants were found to be viable when homozygous with no obvious defects in the adult fly. As Wwox mutant flies were found to exhibit an increased sensitivity to ionising radiation (IR), a number of Wwox proteins specifically deleted or mutated at positions consisting of conserved functional protein motifs, or regions that are highly conserved among WWOX / Wwox homologs. The Wwox variants were tested for their ability to modify the IR sensitivity phenotype. In the course of this study, it was found that background mutations introduced during the generation of the mutant flies was responsible for the IR sensitivity phenotype. As a result, proteomic alterations resulting from changes in Wwox protein levels in Drosophila were investigated in order to ascertain the possible molecular functions of the Wwox protein. 2D-DIGE analysis was conducted on a number of different fly genotypes expressing differing levels of Wwox protein in both adult and embryonic flies. The proteomic changes resulting from lack of Wwox function as well as Wwox over-expression were detected with the proteins of interest identified by mass spectrometry (MS) using both MALDI-TOF/TOF-MS and LC-ESI-MS/MS. Label free quantitative MS analysis was also performed in order to determine the most abundant protein(s) in those spots found to contain multiple proteins. These proteomic studies identified changes in a wide variety of proteins with a significant number of metabolic proteins as well as proteins involved in oxidative stress response as a result of different levels of Wwox expression. Of particular interest, consistent changes in different isoforms of superoxide dismutase 1 (Sod1) were identified. Due to the known roles these proteins play in pro and anti-apoptotic pathways, it is possible that Sod1 and Wwox may work in concert to regulate the delicate balance of defence mechanisms in response to environmental stresses, particularly oxidative stress. The protein/gene targets identified in this work therefore offer some insights into normal Wwox function. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Common fragile sites are a set of genomic locations with a propensity to form lesions, breaks and gaps on mitotic chromosomes upon induction of replication stress. While the exact reasons for their fragility are unknown, CFS display instability in a cell-type specific manner, suggesting a substantial contribution from an epigenetic component. CFSs also overlap with sites of increased breakage and deletions in tumour cells, as well as evolutionary breakpoints, implying that their features shape genome stability in vivo. Previously, factors such as delays in replication timing, low origin density and transcription of long genes have been implicated in instability at CFS locations but comprehensive molecular studies are lacking. Chromatin structure, an important factor that fits the profile of cell-type specific contributor, has also not been investigated yet. Throughout their efforts to determine the factors that lead to the appearance of CFS lesions, investigators have focused on a single component at a time, potentially missing out complex interactions between cellular processes that could underlie fragility. Additional difficulties come from the cell-type specificity of CFS breakage: it indicates that only cell type-matched data would be informative, limiting the scope for studies using publicly available data. To perform a comprehensive study defining the role of different factors in determining CFS fragility, I explored replication timing, transcriptional landscapes and chromatin environment across a number of CFSs in two cell types exhibiting differential CFS breakage. Initially, I characterised the patterns of CFS fragility in the two cell types on both the cytogenetic and the molecular level. I then used a FISH-based technique to investigate the process of mitotic compaction at active CFS sites and found that the cytogenetically fragile core of these sites sits within larger regions which display a tendency to mis-fold in mitosis. The aberrant compaction of these regions could be observed on cytogenetically normal metaphase chromosomes, suggesting that finer scale abnormalities in chromosome structure underlie the cytogenetically visible breaks at fragile sites. I also investigated the links between transcription of long genes and CFS fragility using two approaches: I quantified levels of expression across all fragile sites using RNA-seq and modified transcription at a single active CFS using the CRISPR genome engineering methodology. My results indicate a complex interplay between transcription and CFS fragility: no simple linear correlation can be observed, but an increase of transcriptional levels at the active CFS led to a corresponding increase in fragility. To investigate the influence of the cell type specific replication programme and replication stress on CFS instability, I mapped replication timing genome-wide in unperturbed cells and under conditions of replication stress in both cell types. I found that replication stress induces bi-directional changes in replication timing throughout the genome as well as at CFS regions. Surprisingly, the genomic regions showing the most extreme replication timing alterations under replication stress do not overlap with CFS, implying that CFS instability is not fully explained by replication delays as previously suggested. Instead, I observed a range of replication-stress induced timing changes across CFS regions: while some CFSs appear under-replicated, others display switches to both earlier and later replication as well as differential recruitment of both early and late origins, implying that dis-regulation of replication timing and origin firing, rather than simply delays, underlie the sensitivity to CFS regions to replication stress. Finally, I investigated large-scale chromatin states at two active CFSs throughout S phase and into G2, the cell cycle stages most relevant stage for CFS breakage. I found that changes in large-scale chromatin architecture accompany the replication timing shifts triggered by replication stress, raising the possibility that such alterations contribute to instability. In conclusion, I assessed the influence of multiple relevant factors on CFS fragility. I found that bi-directional replication timing changes and alterations in interphase chromatin structure are likely to play a role, converging to promote mitotic folding problems which ultimately result in the well-described cytogenetic lesions on metaphase chromosomes and genomic instability.

Les Sites Fragiles Communs (SFC) sont des loci incluant des grands gènes où des cassures chromosomiques apparaissent chez des cellules soumises à un stress réplicatif. Il est couramment admis que ces sites sont des régions dont la réplication n’est pas terminée lorsque les cellules entrent en mitose mais le mécanisme sous-jacent restait mal compris. Notre laboratoire a montré par la technique du peignage moléculaire que l’instabilité du site FRA3B chez les lymphocytes est due à la présence d’une grande région pauvre en origines de réplication actives (cœur) dont la réplication ne peut pas se terminer en cas de stress réplicatif. Dans le but de généraliser ces conclusions, j’ai étudié par la technique du « Répli-Seq », le timing de réplication à l’échelle du génome entier chez des cellules lymphoblastoïdes humaines traitées ou non à l’aphidicoline. Les résultats confirment clairement que les SFC connus dans ce type cellulaire correspondent à des régions incluant un grand gène et qui sont déficitaires en réplication sous un stress réplicatif. D’autre part, j’ai montré que retarder l’entrée des cellules en mitose par l’utilisation du RO-3306, un inhibiteur de CDK1, abolit complètement la fragilité des SFC induite par l’aphidicoline chez des lymphocytes et des fibroblastes humains. De plus, j’ai montré que cette suppression des cassures au niveau de FRA3B et FRA16D chez les lymphocytes et de FRA1L chez les fibroblastes ne s’accompagne pas d’une diminution de la transcription de FHIT, WWOX et NEGR1, les trois grands gènes contenus respectivement dans ces trois SFC. Par ailleurs, j’ai corrélé l’expression des gènes FHIT, WWOX, NEGR1 et LSAMP à la fragilité des sites FRA3B, FRA16D, FRA1L et FRA3L dans 7 lignées de fibroblastes et 7 lignées de lymphocytes. J’ai aussi mis en évidence une expression atypique de ces gènes dans certaines de ces lignées et montré que cette expression atypique s’accompagne d’une fragilité atypique du SFC correspondant. Cependant, j’ai montré que l’inhibition de la transcription par deux inhibiteurs différents, le triptolide et l’α-amanitine, ne diminue pas la fragilité induite par l’aphidicoline. Je propose un modèle pour expliquer ces résultats apparemment contradictoires. Enfin, j’ai corrélé la disparition des cassures au niveau de FRA3B dans les cellules traitées à l’aphidicoline et au RO-3306 à une augmentation des événements d’initiation dans la région cœur. J’ai montré que l’inhibition de CDK1 par le RO-3306 permet l’accumulation à la chromatine des facteurs CDC6, CDT1, ORC1 et MCM7 dans les cellules bloquées en phase G2 et que l’accumulation de CDT1 et de CDC6 est indispensable à la diminution de la fragilité sous RO-3306. Je propose un modèle permettant de rendre compte de ces observations qui postule l’existence d’origines de réplication latentes partiellement chargées en complexes de pré-réplication. / Common Fragile Sites (CFS) are loci harboring large genes where chromosome breaks occur in cells under replication stress. It is widely accepted that these sites are regions whose replication is incomplete when cells enter mitosis, but the underlying mechanism remains poorly understood. Our laboratory has shown by the technique of molecular combing, that the instability of FRA3B in lymphocytes is due to the presence of a large region poor in active replication origins (core) whose replication can not be completed under replicative stress. In order to generalize these findings, I studied by the technique of "Repli-Seq", the replication timing across the entire genome in human lymphoblastoid cells treated or not with aphidicolin. The results clearly confirm that CFS known in this cell type correspond to regions including large genes and which are underreplicated under replication stress conditions. Moreover, I have shown that delaying entry of cells into mitosis by using RO-3306, an inhibitor of CDK1, completely abolishes the fragility of SFC induced by aphidicolin in human lymphocytes and fibroblasts. I also have shown that abolition of the breaks at FRA3B and FRA16D in lymphocytes and at FRA1L in fibroblasts is not accompanied by a decreased transcription of FHIT, WWOX and NEGR1, respectively, the three large genes including these three SFC. Moreover, I have correlated the expression of FHIT, WWOX, NEGR1 and LSAMP genes to the fragility of FRA3B, FRA16D, FRA1L and FRA3L sites in 7 lines of fibroblasts and 7 lines of lymphocytes. I also highlighted an atypical expression of these genes in some of these lines and showed that this atypical expression is accompanied by an unusual fragility of the corresponding CFS. However, I have also shown that inhibition of transcription by two different inhibitors, triptolide and α-amanitine, does not diminish the fragility induced by aphidicolin. I propose a model to explain these apparently contradictory results.Finally, I have correlated the disappearance of breaks at FRA3B in cells treated with aphidicolin and RO-3306 with an increase in initiation events in the core region. I have shown that inhibition of CDK1 by the RO-3306 allows the accumulation of factors CDC6 CDT1, ORC1 and MCM7 on the chromatin in cells blocked in G2 phase, and that the accumulation of CDT1 and CDC6 is essential to reducing fragility under RO-3306. I propose a model to explain these observations that postulates the existence of latent replication origins partially loaded with the pre-replication complex.

Les sites fragiles communs (SFC) sont des loci instables en cas de stress réplicatif et des lieux préférentiels de réarrangements dans les tumeurs. Les SFC sont associés aux plus grands gènes du génome et il a été proposé que la transcription de ces gènes soit à l'origine de cassures de l'ADN. Cependant, de nombreux grands gènes transcrits ne sont pas fragiles. Un second modèle, celui de notre laboratoire, associe la fragilité des SFC à un programme de réplication particulier, combinant pauvreté en événements d'initiation et réplication tardive. Il serait alors possible que la transcription soit liée à leur programme de réplication, ce qui conduirait à la fragilité. Pour mieux comprendre ces relations, j'ai modifié la transcription de deux grands gènes et analysé les conséquences sur leur réplication et leur fragilité. Ces manipulations génétiques sont réalisées dans le modèle aviaire DT40, qui permet la modification ciblée d'ADN par recombinaison homologue avec une grande efficacité. De façon surprenante, j'ai observé que la fragilité d'un grand gène est diminuée aussi bien en abolissant sa transcription qu'en l'augmentant. J'ai étudié la densité en événements d'initiation par peignage moléculaire au locus et le programme temporel de réplication dans des clones présentant des niveaux différents de transcription. J'ai ainsi pu montrer que la surexpression massive de deux grands gènes avance le programme temporel de la réplication, les préservant de la fragilité. Au cours de ma thèse, j'ai donc montré que la transcription exerce des effets antagonistes sur la stabilité du génome, bénéfiques ou délétères, selon le niveau d'expression des grands gènes associés aux SFC. / Common Fragile Sites (CFSs) are loci displaying instability upon replicative stress, which localization correlates with chromosomal rearrangements in tumours. CFSs are associated with the largest genes of the genome and it has been proposed that their transcription leads to DNA breaks. However, many transcribed large genes are not fragile. Our laboratory proposed an alternative model in which CFS instability results from a specific replication program, combining late replication with paucity in initiation events. To reconcile the two models, we hypothesized that transcription impacts the replication programs. In order to characterize those potential relationships, I manipulated the transcription of two large genes associated with CFSs and determined the consequences of these manipulations on replication and fragility. I used chicken DT40 cells to perform these analyses because this cellular model allows efficient engineering of specific DNA sequences by homologous recombination. Surprisingly, I observed that increasing or suppressing transcription of large gene both lead to a decrease fragility. I then analyzed clones displaying variable transcription levels. I determined the distribution and density of initiation event, using molecular combing at two loci, as well as the profiles of replication timing along the genes. I showed that a massive overexpression of two large genes led to an earlier replication timing. Overall, my results highlight the opposite effects of transcription on genome stability, which range from beneficial to deleterious depending on the expression level of large genes associated with CFSs.

Les sites fragiles communs (SFCs) sont des régions génomiques particulièrement sensibles au stress réplicatif et sont impliqués dans l’initiation et la progression du cancer. L’Anémie de Fanconi (AF) est une maladie génétique rare qui se caractérise principalement par une aplasie médullaire, des malformations congénitales ainsi qu’une forte prédisposition au cancer chez les patients (leucémies myéloïdes et tumeurs solides de la tête et du coup). L’instabilité génomique a été identifiée comme étant une source majeure de prédisposition des patients AF au cancer et les SFCs sont particulièrement sensibles dans cette maladie. L’AF est causée par la mutation de gènes codant des protéines participant à une voie moléculaire appelée voie FANC qui a été décrite dans la réparation des ponts inter-brins. Malgré l’importance de la voie FANC dans le maintien de la stabilité des SFCs, les mécanismes sous-jacents restent à élucider. Au cours de ma thèse, nous avons identifié un nouveau rôle de FANCD2 dans le maintien des SFCs. En effet, nous montrons que FANCD2 atténue l’expression des gènes présents au sein des SFCs maintenant leur stabilité. De plus, nous montrons que la transcription de ces gènes est nécessaire au recrutement et au rôle de FANCD2 au sein de ces régions. Enfin, nous avons identifié le stress métabolique comme étant un signal induisant l’expression des gènes des SFCs et que FANCD2 module cette réponse. La réduction de ce stress pourrait être une piste thérapeutique intéressante afin de prévenir l’instabilité des SFCs dans l’AF. / Common fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes after replicative stress and promote genomic instability in the earliest steps of tumor development. Proteins involved in replication/repair of CFSs are necessary to prevent their instability. Among them is FANCD2, a key protein of the FANC pathway necessary to resolve inter-strand crosslinks and defective in Fanconi Anemia (FA). FA is a rare genomic instability disorder characterized by bone marrow failure, congenital abnormalities and predisposition to acute myeloid leukemia and epithelial cancer. Genomic instability in FA is supposed to predispose patients to cancers. Importantly, CFSs are more unstable in FA and chromosome breaks observed in FA cells occur preferentially at CFSs. During my PhD, we identified a new role of FANCD2 in CFS stability maintenance. We show that FANCD2 attenuates transcription of the large genes present at CFSs, preventing their instability. Moreover, we demonstrate that transcription is necessary for FANCD2 recruitment and function at CFSs. Importantly, we identified the metabolic stress as a signal triggering CFS gene expression and FANCD2 is necessary to modulate this response. Reducing this stress is a promising therapeutic issue to prevent CFS and genomic instability in FA.

Les Sites Fragiles Communs (SFCs) sont des points de cassures chromosomiques récurrents survenant suite à un stress réplicatif. La majorité d'entre eux ont été identifiés suite à un traitement à l'Aphidicoline (APC), un inhibiteur des ADN polymérases, ce qui explique pourquoi l'étude de leur fragilité est majoritairement basée sur des perturbations de la réplication. Parmi les facteurs impliqués dans la fragilité, ATR (Ataxia Telangiectasia and Rad3 Related), une kinase engagée dans la signalisation des fourches de réplication bloquées, et sa cible CHK1 (Checkpoint Kinase 1), induisent l'apparition de cassures aux SFCs lorsqu'ils sont déplétés dans la cellule. Cependant, ces gènes sont difficiles à étudier car leur déplétion totale est létale pour les cellules. Nous avons donc choisi de développer un modèle basé sur l'utilisation de la lignée MSI de cancer colorectal HCT116de laquelle nous avons isolé des clones haploinsuffisants pour ATR ou CHK1.Ces mutations sont naturelles et sont d'ailleurs dans les cancers MSI. Aujourd'hui, de nouvelles données montrent que la transcription semble aussi être impliquée dans l'induction de certains SFCs. Pour étudier ce mécanisme, nous nous sommes servis de cellules déficientes pour la Topoisomérase 1 (Topo1) grâce à un shARN inductible. Cette protéine est impliquée dans la gestion des interférences entre les machineries de réplication et de transcription et son inhibition est à l'origine d'une forte instabilité chromosomique.Nos études démontrent tout d'abord que l'haploinsuffisance d'ATR ou de CHK1 retrouvée dans les cancers MSI génère des défauts de réplication, des problèmes de checkpoints ainsi que des cassures ADN et en particulier au niveau des SFCs. Une déficience en Topo1 induit aussi l'apparition de problèmes de réplication et une plus grande fragilité ADN, mais elle est aussi capable d'induire des cassures au niveau de certains SFCs, connus pour être sensible à des défauts de réplication. Une étude plus approfondie du SFC le plus fréquemment induit, FRA3B, montre qu'il est sensible à la fois à des défauts dans la voie ATR/CHK1, au stress réplicatif induits par l'aphidicoline mais aussi à la déficience en Topo1. Nos travaux suggèrent que l'haploinsuffisance d'ATR et CHK1 peut favoriser la cancérogenèse à travers l'induction des SFCs et que tous les SFCs n'ont pas les mêmes mécanismes d'induction, laissant la porte ouverte pour l’identification de nouveaux SFCs. / Common Fragile Sites (CFSs) are recurrent chromosomal breakpoints occurring when cells are exposed to replicative stress. Most CFSs have been identified following Aphidicolin (APC) treatment, an inhibitor of DNA polymerase and therefore the working model to explain their fragility is indeed mainly based on perturbation of replication. Among the key actors involved in fragility, ATR (Ataxia Telangiectasia and Rad3 Related), a kinase involved in stalled replication fork signaling, and its target CHK1 (Checkpoint Kinase 1), lead to enhanced chromosome fragility when transiently depleted in cells. However, ATR and CHK1 are difficult to study since their complete depletion is lethal for the cells. We have developed a colorectal cancer HCT116 MSI model harboring ATR or CHK1 haploinsufficient mutations found in MSI cancers. Transcription also seems to be involved in the induction of SFCs. To investigate this mechanism, we created topoisomerase 1 (Topo1) deficient cells with inducible shRNA. This protein is involved in interference between replication and transcription and its inhibition is associated with high chromosomal instability. Our studies indicate that ATR or CHK1 haploinsufficiency found in MSI cancers causes replication and checkpoints defects and induces DNA break particularly at CFSs. Topo1 deficiency is responsible of replication defects and DNA fragility and it induces breaks at some CFSs already known to be sensitive to replication defects. Moreover, a precise study of the most induced CFSs, FRA3B, shows that it is sensitive to defects in ATR/CHK1 pathway, to replicative stress induced by aphidicolin but also to Topo1 deficiency. Our results suggest that ATR and CHK1 haploinsufficiency can promote carcinogenesis through the induction of CFSs. Futhermore, we suggest that all CFSs do not rely on the same induction mechanisms, letting us to postulate that new CFSs are still to be identified.

Submitted by Iara Maluf Tomazella null (iara_tomazella@hotmail.com) on 2017-01-11T17:32:01Z No. of bitstreams: 1 Tese Iara Tomazella - Exemplar definitivo.pdf: 3067128 bytes, checksum: a31fbf1f6b0cc7e28450ded47033bde6 (MD5) / Rejected by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido não contém o certificado de aprovação. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-01-12T16:27:03Z (GMT) / Submitted by Iara Maluf Tomazella null (iara_tomazella@hotmail.com) on 2017-01-17T17:22:25Z No. of bitstreams: 1 Tese Iara Tomazella repositório.pdf: 3251616 bytes, checksum: ae9f89d671c6cbd6a55d8e1f7d64acea (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-18T19:17:25Z (GMT) No. of bitstreams: 1 tomazella_im_dr_jabo.pdf: 3251616 bytes, checksum: ae9f89d671c6cbd6a55d8e1f7d64acea (MD5) / Made available in DSpace on 2017-01-18T19:17:25Z (GMT). No. of bitstreams: 1 tomazella_im_dr_jabo.pdf: 3251616 bytes, checksum: ae9f89d671c6cbd6a55d8e1f7d64acea (MD5) Previous issue date: 2016-12-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Mazama gouazoubira (2n=70; NF=70), popularmente chamado de veado-catingueiro, é conhecido por apresentar fragilidade cromossômica, responsável pela variação cromossômica intraespecífica, caracterizada pela presença de translocações Robertsonianas e cromossomos B. Não existem dados sobre a localização das regiões cromossômicas envolvidas com os rearranjos em M. gouazoubira e com a possível existência de sítios frágeis (SFs) nos pontos em que ocorrem esses rearranjos. Assim, torna-se necessário avaliar o polimorfismo cromossômico apresentado pela espécie e identificar os SFs, investigando sua relação com o polimorfismo. Dos 135 animais analisados, 68 (50,37%) são individuos variantes, 47 animais (69,12%) apresentaram cromossomos B, seis animais (8,82%) são heterozigotos para uma translocação Robertsoniana, um indivíduo (1,47%) é homozigoto para uma translocação Robertsoniana, 14 animais (20,59%) são portadores de cromossomos B e heterozigotos para uma translocação Robertsoniana. Foram identificados sete tipos distintos de translocações (X;16, X;21, 7;21, 8;21, 4;16, 20;26, 14;16), envolvendo nove cromossomos diferentes. As translocações X-autossômicas foram confirmadas pelas técnicas de banda C, coloração Ag-RON, hibridização in situ fluorescente (FISH) com sondas teloméricas e pintura cromossômica com a sonda específica do cromossomo X. Foi observada uma grande variabilidade de cromossomos B entre os indivíduos analisados, sendo esses cromossomos altamente heterogêneos em relação aos padrões de distribuição de heterocromatina, presença e quantidade de rDNA nas regiões organizadores de nucléolos (RON), localização de sequências teloméricas e homologias entre lotes A e B. A afidicolina foi um eficiente indutor de sítios frágeis comuns (SFCs), revelando a ocorrência de SFCs na forma de “gaps” e quebras, tanto cromatídicas como cromossômicas. A técnica de banda G localizou 531 SFCs distribuídos em 18 pares cromossômicos (X, 1, 2, 4, 5, 6, 7, 8, 10, 11, 13, 14, 16, 17, 18, 21, 22 e 34), sendo que a maioria está localizada em pontos de transição entre as bandas claras e as bandas escuras. As diferentes taxas de SFCs apresentada por cada cromossomo mostrou que alguns pares cromossômicos são mais frágeis do que outros. Dos 18 pares cromossômicos com SFCs, sete estão relacionados com as translocações Robertsonianas observadas no veado-catingueiro e somente um cromossomo envolvido no polimorfismo não possui SFCs. Assim, o polimorfismo cromossômico apresentado pelo M. gouazoubira pode estar relacionado com a fragilidade cromossômica. É necessário aprofundar os estudos para entender qual o impacto desse polimorfismo na população brasileira do veado-catingueiro. / Mazama gouazoubira (2n = 70; FN = 70), popylarly known as brown brocket deer, is known to have chromosomal fragility, which is responsible for intraspecific chromosome variation, characterized by the presence of Robertsonian translocations and B chromosomes. There are no data of the location of the chromosome regions involved in rearrangements of M. gouazoubira and the possible existence of fragile sites (FSs) in points where breaks occur. Thus, it is necessary to evaluate the chromosomal polymorphism presented by this species and to identify the FSs, investigating the relationship between FSs and polymorphism. Were analyzed 135 animals, of which 68 (50.37%) were variant individuals, 47 animals (69.12%) had B chromosomes, six animals (8.82%) were heterozygous for a Robertsonian translocation, one individual (1.47%) was homozygous for a Robertsonian translocation, 14 animals (20.59%) presented both B chromosomes and heterozygotes for a Robertsonian translocation. Were identified seven different types of translocations (X;16, X;21, 4;16, 14;16, 7;21, 20;26, 8;21) involving nine different chromosomes. X-autosomal translocations were confirmed by C-banding, Ag-NOR staining, Fluorescence in situ hybridization (FISH) with telomeric probes and chromosome painting with X chromosome-specific probe. A large variability of B chromosomes was observed among the analyzed individuals. These chromosomes were highly heterogeneous in relation to pattern of heterochromatin distribution, presence and amount of rDNA in nucleolar organizer region (NOR), lozalization of telomeric sequences and homologies between chromosome complements A and B. Aphidicolin was an efficient inducer of common fragile sites (CFSs), showing the occurrence of CFSs in gaps and breaks, both chromatid and chromosomal. The G-banding located 531 CFSs distributed in 18 chromosome pairs (X, 1, 2, 4, 5, 6, 7, 8, 10, 11, 13, 14, 16, 17, 18, 21, 22 and 34). It was found that the most CFSs are localized at the boundaries between the bright bands and dark bands. The different rates of CFSs presented by each chromosome showed that some chromosome pairs are more fragile than others. Of the 18 chromosomes pais with CFSs, seven are related to the Robertsonian translocations observed in brown brocket deer, and only one chromosome involved with polymorphism does not have CFSs. Thus, the chromosomal polymorphism presented by M. gouazoubira may be related to chromosomal fragility. It is necessary to deepen the studies to understand the impact of this polymorphism on the Brazilian population of brown brocket deer. / FAPESP: 2013/06100-7

No description available.

Im ersten Teil dieser Arbeit wurden mittels molekular-zytogenetischer Methoden die chromosomalen Aberrationen in c-myc aktivierten ARFnull- und INK4a/ARFnull-Mauslymphomen untersucht. Die zytogenetischen Ergebnisse wurden mit dem Therapieverlauf der Mäuse nach Cyclophosphamid-Behandlung verglichen. In den ARFnull-Lymphomen erkannten wir den Gewinn des Chromosoms 14 als einen Marker für gute und den Gewinn des Chromosoms 6 als Marker für schlechte Behandlungserfolge. Auf den Chromosomen 6 und 14 der Maus liegen demnach bisher unbekannte Gene, welche für die Wahl der Behandlungsmethode ARF-defizienter Tumore von entscheidender Bedeutung sind. Der zweite Teil der Arbeit befaßt sich mit Common Fragile Sites (CFS), die als "hot spots" für chromosomale Brüche und Umbauten in Tumorgenese und Karyotypevolution diskutiert werden. CFSs treten als seltene Lücken im Chromatin oder als partiell deletierte bzw. rearrangierte Chromosomen auf. Ihre Zahl wird durch Zugabe von Replikations-hemmenden Chemikalen, wie Aphidicolin (APC), erheblich gesteigert. Wir untersuchten die CFS-Expression in Lymphozytenkulturen der Mausstämme BALB/c und C57BL/6. Die APC-induzierten CFS-Häufigkeiten der Chromosomenbanden wiesen im Vergleich zwischen beiden Mausstämmen eine signifikante Korrelation und damit eine starke Konservierung auf. Ebenfalls wurde zwischen Maus / Mensch-syntenischen Bereichen eine Konservierung der CFS-Häufigkeiten detektiert. Die Tendenz zur CFS-Bildung ist also ein spezifisches Merkmal jedes chromosomalen Abschnitts, das evolutionär konserviert ist. Weiterhin wurden einzelne CFSs mit molekular-zytogenetischen Methoden genauer kartiert. Auf diese Weise beschrieben wir erstmals eines der zehn häufigsten CFSs menschlicher Lymphozyten, FRA7K, und erweiterten somit die Anzahl molekular charakterisierter humaner CFSs auf insgesamt 13. Für fünf dieser CFSs analysierten wir mittels FISH-Analyse die homologen Bereiche im Mausgenom hinsichtlich ihrer CFS-Expression. Die beobachteten Läsionen traten jeweils in exakt den entsprechenden Sequenzen auf. Vereint man diese fünf Beispiele (FRA2G/Fra2D, FRA7G/Fra6A3.1, FRA7H/Fra6B1, FRA7K/Fra12C1 und FRA9E/Fra4C2) mit bekannten Homologen aus der Literatur, so wurde für insgesamt acht CFSs eine Konservierung zwischen Mensch und Maus auf molekularer Ebene gefunden. Trotz zahlreicher Untersuchungen ist der zelluläre Mechanismus, der die Ausbildung von CFSs an spezifischen Stellen des Genoms bewirkt, bis heute weitgehend unklar. Bekannt ist, daß CFSs durch Replikationsinhibition induziert werden und in Metaphase-Zellen sichtbar werden, welche DNA-Replikations-Checkpoints unterlaufen haben.Sämtliche jüngeren Studien beschrieben Inseln erhöhter DNA-Helix-Flexibilität (Schwankungen im Biegungswinkel des Moleküls) in den Bereichen von CFSs. Wir berechneten die DNA-Helix-Flexibilität entlang der Sequenz für alle molekular kartierten humanen und Maus-CFSs sowie für stabile Kontrollregionen. Anders als in der Literatur beschrieben, fanden wir für Mensch und Maus, daß sich CFSs und Kontroll-DNAAbschnitte in ihrer Dichte an Inseln mit erhöhter DNA-Helix-Flexibilität nicht unterschieden. Nahezu alle Regionen der häufigen molekular charakterisierten CFSs umfassen große Gene, deren Exons mehr als 650 kb genomische Sequenz überspannen. Im Gegensatz dazu traten große Gene in den stabilen Kontrollregionen deutlich seltener auf. Öffentlich zugängliche RNA-Expressionsdaten dieser Gene zeigten, daß CFSs bevorzugt in Bereichen mit transkriptionell aktiven, großen Genen entstehen. Darauf und auf dem Wissen aus der Literatur begründen wir die Hypothese, daß eine Blockierung der DNAReplikationsgabel, vor allem in Bereichen großer transkriptionell aktiver Gene - eventuell begründet durch deren offenere Chromatinstruktur - und das anschließende Unterlaufen von Zellzyklus-Checkpoints an der Bildung von CFSs und damit in einem Anstieg von Doppelstrangbrüchen beteiligt sind.