No description available.

Understanding chromosome structure and genome organization of Culicine mosquitoes can potentially contribute to the development of novel approaches to vector control. However, because of highly repetitive nature of the Aedes and Culex genomes, the structure of their polytene chromosomes is damaged by ectopic contacts that make the analysis difficult. Mitotic chromosomes from imaginal discs of 4th instar larvae of Aedes aegypti were tested as a source for the physical genome mapping for this mosquito. Chromosomes in imaginal discs are 10 times more abundant than chromosomes in nervous ganglia, and they do not accumulate chromosomal mutation as cell line chromosomes do. Prometaphase chromosomes in imaginal discs of Ae. aegypti are 4-5 times longer than metaphase chromosomes and can provide higher resolution for physical mapping. Cold temperature (+16°C) was proven to increase the number of the chromosomes. Hypotonic solution treatment of live larvae was proven to elongate chromosomes and improve banding patterns. We differentially stained these mitotic chromosomes with Giemsa and YOYO-1 to revile the banding pattern. We applied fluorescent in situ hybridization (FISH) procedure developed for human chromosomes to Ae. aegypti chromosomes. A strain from Culex pipiens, Cx. quinquefasciatus and their hybrids from the natural population in Virginia was successfully colonized in the laboratory. This strain can be used as a reliable source for cytogenetic studies. / Master of Science in Life Sciences

No description available.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Changes in chromosome number and structure may cause serious diseases. Cytogenetic tests leadin to set of karyotype are done for detecting these abnormalities. Chromosomes are visualised with proper methods and karyotype is made up most often. Manual karyotyping is time-consuming and expensive task. Because of this, researchers have been developing automated karyotyping systems. Karyotyping systems classify chromosomes into classes based on their characteristic features. Overlapping and bent chromosomes are limitations for automatic classification since they ocur at almost every mitosis. Accuracy and reliability of karyotyping systems still depend on the human intervention. Overcoming of these problems and development of fully automated system is the aim of modern approaches.

(ENGLISH) Ubiquitin signaling is a key regulatory mechanism for many important cellular processes such as transcription, differentiation and cell division. Cell division requires duplication of all genetic material during S-phase followed by its precise partitioning between two daughter cells during mitosis. Misregulation of the complex mitotic machinery may lead to aneuploidy and genomic instability, known drivers of tumorigenesis. Indeed, systematic genetic analysis of many cancer tissues over the last decades, indicates the presence of severe chromosome abnormalities in thousands of cancer tissue samples. In this work, I investigated the function of two components of ubiquitin signaling, the deubiquitinating enzyme UCHL3 and the E3 ubiquitin ligase TRIM15. The hypothesized role of E3 ligase TRIM15 in the cell cycle regulation could not be confirmed by our experiments, but I observed an effect on cell adhesion and motility instead. UCHL3 was identified using high-content visual siRNA screen, as a critical factor controlling genome segregation and integrity. Interestingly, it has been previously reported that UCHL3 levels are altered in various cancer types, especially colon cancer. My data demonstrate that UCHL3 drives proper alignment of chromosomes at the metaphase plate by facilitating...

La plupart des espèces bactériennes possèdent un chromosome circulaire qui est répliqué de façon bidirectionnelle au cours du cycle cellulaire. Bien que la réplication et la ségrégation du chromosome bactérien se développent simultanément, la ségrégation du chromosome s’accomplit après la fin de la réplication et avant la fermeture du septum. La circularité du chromosome bactérien et le grand nombre d’événements de recombinaison homologue donnent lieu à la création des dimères de chromosome dans une fraction de la population cellulaire. Chez Escherichia coli et Bacillus subtilis, la dimérisation des chromosomes se produit respectivement dans 15% et 25% des cas. Un chromosome dimérique doit être résolu avant la fermeture du septum. Chez les espèces bactériennes les plus étudiées, les chromosomes dimériques sont résolus par un système de recombinaison site spécifique hautement réservé incluant deux recombinases à tyrosine, XerC et XerD, et un site génomique dans la région terminus du génome bactérien, appelé le site dif (deletion induced filamentation). L’organisation spatio-temporelle du système de recombinaison site spécifique Xer/dif et l’activation de ce dernier sont réglementées par la protéine transmembranaire impliquée dans la division cellulaire, FtsK. D’autre part, des études récentes ont mis en évidence l’existence de plusieurs éléments mobiles appelés IMEXs (Integrative Mobile Elements Exploiting Xer), capables d’exploiter le système Xer/dif pour leur intégration dans le génome bactérien. Chez E. coli, des déficiences dans la résolution des dimères de chromosome se terminent par le guillotinage du chromosome dimérique au cours de la division cellulaire, ce qui entraîne l’induction de la réponse SOS chez les cellules filles et la mort de ces dernières. Dans cette thèse, le site dif a été identifié et caractérisé chez Caulobacter par une combinaison d’approches in vivo et in vitro. Fait intéressant, il a été démontré que chez Caulobacter, contrairement à E. coli, la perturbation du système Xer/dif ne mène pas au guillotinage du chromosome, et les cellules portant un système Xer/dif défectueux contourne cette déficience en adoptant un nouveau mode de cycle cellulaire. De plus, notre analyse comparative entre les terminus des souches sauvages de C. crescentus a également permis de révéler la présence d’un IMEX putatif de 71 kb dans le terminus de C. crescentus NA1000. / Most bacteria possess a single circular chromosome which is replicated bidirectionally during the cell cycle. Although replication and segregation of the chromosome in bacteria develops simultaneously, the segregation of the chromosome occurs after the completion of replication and before the closure of the septum. The circularity of the bacterial chromosome and the high number of homologous recombination events that occur during replication result in the creation of chromosome dimers in a fraction of the cell population. In Escherichia coli and Bacillus subtilis, chromosome dimer formation occurs, respectively, in 15% and 25% of the cell population during replication, which needs to be resolved before the closure of division septum. In most of the well-studied bacterial species, chromosome dimers are resolved by a highly conserved site-specific recombination system which employs two tyrosine recombinases, XerC and XerD, and a recombination genomic site located in the terminus region of the bacterial chromosome called dif (deletion induced filamentation). The temporo-spatial organization of the Xer/dif site-specific recombination system, along with its activation, is regulated by a cell division transmembrane protein, FtsK. In E. coli, deficiencies in the resolution of chromosome dimers result in the guillotining of the dimeric chromosome during the cell division leading to the continuous induction of SOS response in the daughter cells and the death of the latter ones. In my thesis, the dif site in Caulobacter is identified and characterized by a combination of in vitro and in vivo approaches. Interestingly, it was observed that, unlike E. coli, in Caulobacter perturbations in the chromosome dimer resolution system do not result in the guillotining of the chromosome dimers. Instead, Caulobacter cells bearing deficiencies in the resolution of dimeric chromosomes adopt a new mode of cell cycle to bypass this deficiency.

No description available.

La cytogénétique est une branche de la génétique qui étudie les chromosomes et leur intégrité ainsi que les conséquences de leur transmission et de leur expression. Plusieurs syndromes et maladies ont pu être expliqués par cette discipline. Certaines anomalies chromosomiques de structure ont d'ailleurs contribué à identifier des gènes, des fonctions de gènes ou à caractériser des structures chromosomiques. Dans cet ordre d'idées, nous avons utilisé deux isodicentriques du bras long du chromosome Y (idic(Y)(p11.3)) pour étudier la fonction d'une protéine du kinétochore, CENP-B, dans le mécanisme d'inactivation de kinétochore. Pour ce faire, nous avons premièrement fait une caractérisation cytogénétique des deux idic(Y)(p11.3) à l'aide des techniques de cytogénétique classique et de cytogénétique moléculaire. Nous avons ainsi déterminé approximativement le point de cassure des deux isodicentriques, soit en Yp11.3. Comme les deux chromosomes avaient une structure très semblable, mais que les patients présentaient un phénotype clinique très différent, nous avons investigué les niveaux de mosaïcisme dans différents tissus chez les deux patients. Il est connu qu'un chromosome possédant deux centromères capables de former le kinétochore peut être très instable lors des divisions cellulaires. Ainsi, la cellule a mis au point un mécanisme permettant d'inactiver un des kinétochores du chromosome dicentrique. Une récente revue a proposé que la protéine CENP-B jouerait un rôle dans ce mécanisme. Cependant, comme le chromosome Y ne possède pas la séquence d'ADN liant cette protéine, il était intéressant de vérifier si une inactivation des kinétochores avait eu lieu dans nos idic(Y)(p11.3). À l'aide d'un anticorps dirigé contre la protéine CENP-C, connue comme un marqueur de kinétochore actif, nous avons montré que plus de 40% des chromosomes dicentriques avaient subi une inactivation d'un de leur kinétochore. Enfin, la présence de la protéine CENP-B dans ces kinétochores a été étudiée. Nous avons montré que la protéine CENP-B était présente à tous les autres centromères, sauf ceux de l'idic(Y)(p11.3). Ainsi, nous proposons que la protéine CENP-B n'est pas impliquée directement dans le mécanisme d'inactivation de kinétochore du chromosome Y. Par contre, nous ne pouvons pas exclure qu'elle joue un rôle indirect, soit par une interaction protéine/protéine, soit à une étape en amont dans le mécanisme d'inactivation.

The chromosome constitution of human cleavage stage embryos has been extensively investi-gated using a variety of techniques, revealing high levels of aneuploidy and mosaicism. However, the final phase of preimplantation development, the blastocyst stage has received relatively little attention mostly because it is only recently that embryo culture has become sufficiently well optimised to reliabley generate blastocysts. One of the aims of this study was to examine blastocyst cytogenetics, characterising the extent and variety of aneuploidy and, where possible, determining the origin of the abnormalities detected. Both the frequency of aneuploidy and the incidence of mosaicism were significantly lower in the 52 embryos generated by 20 patients that had successfully undergone the first cellular differentiation, producing trophectoderm (TE) and inner cell mass (ICM). Valuable tools for the detailed chromosomal analysis of blastocysts, used in both research and clinical contexts, were comparative genomic hybridization (CGH) and array CGH (aCGH). However, validation of these methods, especially aCGH, was required in order to verify accuracy. A low error rate and a low misdiagnosis risk were demonstrated. The morphology of 1397 embryos at the cleavage and blastocyst stages from 229 patients was evaluated in relation to their chromosomal complement. The results obtained during this part of the project showed that, in general, there is little correlation between cleavage stage morphology and chromosome status. A weak link between morphology and aneuploidy, however, was found for embryos at the blastocyst stage. Chromosomally normal female embryos had a tendency to grow faster than male embryos at the cleavage stage and therefore tended to achieve superior morphological scores, whereas the trend was reversed at the blastocyst stage. Abnormal embryos carrying types of aneuploidy compatible with formation of a clinically recognised pregnancy had morphologies indistinguishable from those of euploid embryos. This study also aimed to utilise aCGH for the preimplantation genetic diagnosis (PGD) of imbal-ances due to structural chromosome rearrangements (e.g. translocations) in 39 carriers, a total of 139 embryos were assessed. The data obtained revealed that carriers of Robertsonian translocations are at increased risk of aneuploidy affecting additional chromosomes not involved the translocation, a phenomenon known as an interchromosomal effect (ICE). Finally, the clinical outcomes of 300 patients undergoing preimplantation genetic screening (PGS) using aCGH, for various different indications, were evaluated at both the cleavage (795 embryos) and blastocyst stages (1097 embryos). The pregnancy rate following cleavage stage biopsy was significantly lower than following blastocyst stage biopsy. The miscarriage rate was significantly reduced following PGS for patients with recurrent miscarriages. This work provided promising data supporting the clinical use of comprehensive chromosome analysis for the screening or diagnosis of preimplantation embryos and also yielded scientifically useful information concerning the frequency and nature of aneuploidy at the final stage of development before implantation.