The World Health Organization estimates that vector-borne diseases account for 17% of the global burden of all infectious diseases and has identified the mosquito as the most dangerous of all disease-transmitting insects, being responsible for several million deaths and hundreds of millions of cases each year. The study of mosquito genomics provides a deeper understanding of the molecular mechanisms involved in every aspect of vector biology, such as sex determination, host-parasite interaction, ecology, feeding behavior, immunity and evolutionary trends and can be used for the development of new strategies for vector control. We developed the first map of the mitotic chromosomes of the major vector for West Nile fever and lymphatic filariasis, Culex quinquefasciatus. The map was then successfully utilized for mapping of approximately 90% of available genetic markers to their precise positions on the chromosomes. Idiograms were integrated with 140 genetic supercontigs representing 26.5% of the genome. A linear regression analysis demonstrated good overall correlation between the positioning of markers on physical and genetic linkage maps. This will improve gene annotation and help in distinguishing potential haplotype scaffolds and regions of segmental duplications. It will also facilitate identification of epidemiologically important genes that can be used as targets for the vector control and provide a better framework for comparative genomics that will help understanding of the evolution of epidemiologically important traits. In another study, we confirmed the presence of the newly described species, Anopheles daciae, in regions of Russia using molecular data. Although sympatric with its sibling species, Anopheles messeae, five nucleotide substitutions in the internal transcribed spacer 2 of ribosomal DNA can be used to distinguish the morphologically similar species. Chromosome rearrangements have a significant impact on mosquito adaptation and speciation. Using sequencing data in combination with karyotyping, we demonstrated that significant differences in inversion frequencies distinguish An. messeae from An. daciae, suggesting that these inversions are actively involved in adaptation and speciation. It is essential to have reliable toolbox for correct identification of these species and to know their range for future possible malaria outbreaks prevention. / Ph. D.

La présence de cassures double-brin de l'ADN en mitose est problématique pour les cellules, car cette situation produit des fragments de chromosome ne possédant pas de centromères. En l'absence d'un mécanisme permettant leur prise en charge, ces fragments acentriques n'étant pas attachés au fuseau mitotique, pourraient être ségrégés aléatoirement dans les cellules filles, causant de l'instabilité génomique. Nous avons découvert un mécanisme permettant la transmission correcte des fragments acentriques dans les cellules filles via une structure faisant le lien entre les deux fragments cassés. Plusieurs protéines sont recrutées sur les cassures, comme les kinases mitotiques BubR1 et Polo, et favorisent la ségrégation correcte de ces chromosomes cassés. Cependant, les mécanismes permettant le recrutement de BubR1 et Polo sur les cassures d'ADN en mitose sont inconnus. De plus, les mécanismes moléculaires par lesquels BubR1 et Polo favorisent la ségrégation correcte des fragments acentriques restent à être identifiés. La première partie de mon projet a été d'étudier le rôle et la régulation de BubR1 sur les cassures d'ADN pendant la mitose. Nous avons montré que BubR1 requiert Bub3 pour se localiser sur les chromosomes cassés afin de favoriser leur ségrégation correcte. Nous avons également détecté l'accumulation de FizzyCDC20, un cofacteur de l'E3 ubiquitine ligase APC/C (Anaphase-Promoting- Complex/Cyclosome), sur les cassures d'ADN, et son recrutement dépend de son interaction avec la KEN Box de BubR1. De plus, l'utilisation d'un substrat synthétique de l'APC/C nous a permis de démontrer que la dégradation par l'APC/C est inhibée localement autour du chromosome cassé, de manière dépendante de BubR1. Ces résultats suggèrent fortement que le complexe BubR1/Bub3 recrut é sur les cassures d'ADN inhibe localement l'APC/C en séquestrant FizzyCDC20 et empêche ainsi la dégradation de substrats clefs impliqués dans la ségrégation correcte des chromosomes cassés. La seconde partie de mon projet a été d'étudier les relations d'interdépendance entre Polo et BubR1/Bub3/Fizzy sur les cassures d'ADN en mitose. Nous avons utilisé un laser UV pulsé pour induire des cassures dans un chromosome à un instant précis pendant la mitose, puis nous avons suivi le recrutement de protéines tagguées GFP sur les cassures de chromosome. Cette étude révèle que Polo est rapidement recrutée sur les cassures d'ADN et précède BubR1, Bub3 et Fizzy. De plus, la disparition de BubR1, Bub3 et Fizzy des cassures d'ADN coïncide avec la télophase alors que Polo disparait des cassures pendant l'interphase. Nous avons également montré que le recrutement de BubR1, Bub3 et Fizzy sur les cassures d'ADN est retardé dans les mutants polo, indiquant que Polo est requis pour un recrutement efficace de BubR1, Bub3 et Fizzy sur les cassures d'ADN. Pour finir, nous avons montré que l'accumulation de Polo et BubR1/Bub3/Fizzy sur les cassures d'ADN dépend de deux composants de la réponse aux dommages à l'ADN, le complexe MRN (Mre11-Rad50-Nbs1) et ATM (ataxia-telangiectasia mutated). Ce travail a permis d'avoir une meilleure compréhension sur la dynamique de recrutement de Polo et BubR1/Bub3/Fizzy sur les cassures d'ADN en mitose. De plus, le mécanisme moléculaire par lequel le complexe BubR1/Bub3 agit pour faciliter la ségrégation des chromosomes cassés a pu être en partie élucidé. / The presence of DNA double strand breaks (DSB) during mitosis is challenging for the cell, as it produces fragments of chromosome lacking a centromere. If not processed, this situation can cause genomic instability resulting in improper segregation of the broken fragments into daughter cells. We uncovered a mechanism by which broken chromosomes are faithfully transmitted to daughter cells via the tethering of the two broken chromosome ends. Several proteins including the mitotic kinase BubR1 and Polo are recruited to the breaks and mediate the proper segregation of the broken fragments. However, the mechanism underlying Polo and BubR1 recruitment to DNA breaks is unknown. Moreover, the molecular mechanisms by which Polo and BubR1 mediate the proper segregation of the broken fragments remain to be elucidated. We first investigated the role and regulation of BubR1 on DNA breaks during mitosis. We show that BubR1 requires Bub3 to localize on the broken chromosome fragment and to mediate its proper segregation. We also find that FizzyCdc20, a co--‐factor of the E3 ubiquitin ligase Anaphase--‐Promoting--‐Complex/Cyclosome (APC/C), accumulates on DNA breaks in a BubR1 KEN box--‐dependent manner. A biosensor for APC/C activity demonstrates a BubR1--‐dependent local inhibition of APC/C around the segregating broken chromosome. These results are consistent with a model where Bub3/BubR1 complex on DNA breaks functions to inhibit the APC/C locally via the sequestration of FizzyCdc20, thus preserving key substrates from degradation, which promotes proper transmission of broken chromosomes. In a second study, we investigated the dependency relationship between Polo and BubR1/Bub3/Fizzy on DNA breaks in mitosis. We used a pulsed UV laser to break one chromosome at a define time during mitosis. We immediately follow the recruitment of GFP--‐tagged proteins to laser--‐induced DNA breaks. My study reveals that Polo is promptly recruited to DNA breaks and precedes BubR1, Bub3 and Fizzy. In addition, while BubR1, Bub3 and Fizzy dissociation from the breaks coincide with telophase and the nuclear envelope reformation, Polo remains on the breaks well into interphase. We further show that the appearance of BubR1, Bub3 and Fizzy on DNA breaks is delayed in polo mutant, indicating that Polo is required for the robust and efficient recruitment of BubR1, Bub3 and Fizzy to DNA breaks. Finally, the timely accumulation of Polo, BubR1 and Bub3 to DNA breaks depends on two components of the DNA Damage Response, the MRN complex (Mre11--‐Rad50--‐Nbs1) and ATM (ataxia--‐telangiectasia mutated). This work gives us a better understanding on how Polo and BubR1, Bub3 and FizzyCdc20 are recruited to DNA breaks in mitosis and how they promote broken chromosomes segregation.

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Multicolor fluorescence in-situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Often, the intensity distributions between different images are found to be considerably different and the difference becomes the source of misclassifications of the pixels. Improved pixel classification accuracy is the most important task to ensure the success of the M-FISH technique. Along with a reliable pixel classification method, automation of the karyotyping process is another important goal. The automation requires segmentation of chromosomes, which not only involves object/background separation but also involves separating touching and overlapping chromosomes. While automating the segmentation of partially occluded chromosomes is an extremely challenging problem, a pixel classification method that satisfies both high accuracy and minimum human intervention has not been realized. The main contributions of this dissertation include development of a new feature normalization method for M-FISH images that reduces the difference in the feature distributions among different images, and development of a new decomposition method for clusters of overlapping and touching chromosomes. A significant improvement was achieved in pixel classification accuracy after the new feature normalization. The overall pixel classification accuracy improved by 40% after normalization. Given a cluster, a number of hypotheses was formed utilizing the geometry of a cluster, pixel classification results, and chromosome sizes, and a hypotheis that maximized the likelihood function was chosen as the correct decomposition. Superior decomposition results were obtained using the new method compared to the previous methods. Contributions also include development of a color compensation method for combinatorially stained FISH images (including M-FISH images) based on a new signal model for multicolor/multichannel FISH images. The true signal was recovered based on the signal model after color compensation. The resulting true signal does not have color spreading (channel crosstalk) among different color channels. Two new unsupervised nonparametric classification methods for M-FISH images are also introduced in this dissertation: a fuzzy logic classifier and a template matching method (a minimum distance classifier). While both methods produce an equivalent accuracy compared to a supervised classification method, their computation time is significantly less than a Bayes classifier. Highly sophisticated and practical algorithms have been developed through this research. Using the developed methods, the amount of human intervention required will be significantly reduced: chromosomes are reliably and accurately segmented from the background, pixels are accurately classified, and clusters of overlapping and touching chromosomes are automatically decomposed. / text

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