During the last decades, mitochondria and their DNA have become a hot topic of research due to their essential roles which are necessary for cells survival and pathology. In this study, multiple methods have been developed to help with the understanding of mitochondrial DNA and its evolution. These methods tackle two essential problems in this area: the accurate annotation of protein-coding genes and mitochondrial genome rearrangements. Mitochondrial genome sequences are published nowadays with increasing pace, which creates the need for accurate and fast annotation tools that do not require manual intervention. In this work, an automated pipeline for fast de-novo annotation of mitochondrial protein-coding genes is implemented. The pipeline includes methods for enhancing multiple sequence alignment, detecting frameshifts and building protein profiles guided by phylogeny. The methods are tested on animal mitogenomes available in RefSeq, the comparison with reference annotations highlights the high quality of the produced annotations. Furthermore, the frameshift method predicted a large number of frameshifts, many of which were unknown. Additionally, an efficient partially-local alignment method to investigate genomic rearrangements in mitochondrial genomes is presented in this study. The method is novel and introduces a partially-local dynamic programming algorithm on three sequences around the breakpoint region. Unlike the existing methods which study the rearrangement at the genes order level, this method allows to investigate the rearrangement on the molecular level with nucleotides precision. The algorithm is tested on both artificial data and real mitochondrial genomic sequences. Surprisingly, a large fraction of rearrangements involve the duplication of local sequences. Since the implemented approach only requires relatively short parts of genomic sequence around a breakpoint, it should be applicable to non-mitochondrial studies as well.

In a recent article, Bai and Perron (2003, Journal of Applied Econometrics) present a comprehensive discussion of computational aspects of multiple structural change models along with several empirical examples. Here, we report on the results of a replication study using the R statistical software package. We are able to verify most of their findings; however, some confidence intervals associated with breakpoints cannot be reproduced. These confidence intervals require computation of the quantiles of a nonstandard distribution, the distribution of the argmax functional of a certain stochastic process. Interestingly, the difficulties appear to be due to numerical problems in GAUSS, the software package used by Bai and Perron. / Series: Research Report Series / Department of Statistics and Mathematics

L'augmentation du nombre de génomes totalement séquencés rend de plus en plus efficace l'étude des mécanismes évolutifs à partir de la comparaison de génomes contemporains. L'un des principaux problèmes réside dans la reconstruction d'architectures de génomes ancestraux plausibles afin d'apporter des hypothèses à la fois sur l'histoire des génomes existants et sur les mécanismes de leur formation. Toutes les méthodes de reconstruction ancestrale ne convergent pas nécessairement vers les mêmes résultats mais sont toutes basées sur les trois mêmes étapes : l'identification des marqueurs communs dans les génomes contemporains, la construction de cartes comparatives des génomes, et la réconciliation de ces cartes en utilisant le critère de parcimonie maximum. La qualité importante des données à analyser nécessite l'automatisation des traitements et résoudre ces problèmes représente de formidables challenges computationnels. Affiner le modèles et outils mathématiques existants par l'ajout de contraintes biologiques fortes rend les hypothèses établies biologiquement plus réalistes. Dans cette thèse, nous proposons une nouvelle méthode permettant d'identifier des marqueurs communs pour des espèces évolutivement distantes. Ensuite, nous appliquons sur les cartes comparatives reconstituées une nouvelle méthode pour la reconstruction d'architectures ancestrales basée sur les adjacences entre les marqueurs calculés et les distances génomiques entre les génomes contemporains. Enfin, après avoir corrigé l'algorithme existant permettant de déterminer une séquence optimale de réarrangements qui se sont produits durant l'évolution des génomes existants depuis leur ancêtre commun, nous proposons un nouvel outil appelé VIRAGE qui permet la visualisation animée des scénarios de réarrangements entre les espèces / Abstract

In a recent article, Bai and Perron (2003, Journal of Applied Econometrics) present a comprehensive discussion of computational aspects of multiple structural change models along with several empirical examples. Here, we report on the results of a replication study using the R statistical software package. We are able to verify most of their findings; however, some confidence intervals associated with breakpoints cannot be reproduced. These confidence intervals require computation of the quantiles of a nonstandard distribution, the distribution of the argmax functional of a certain stochastic process. Interestingly, the difficulties appear to be due to numerical problems in GAUSS, the software package used by Bai and Perron. / Series: Research Report Series / Department of Statistics and Mathematics

As alterações cromossômicas estruturais associadas a fenótipos clínicos oferecem a oportunidade de identificação e localização de genes cujas mutações possam estar determinando essas patologias, tendo em vista a possibilidade de que esses genes podem ter sido alterados pelas quebras ou ter o número de cópias modificado. Um número cada vez maior de evidências aponta para a participação de certas seqüências do genoma na formação de rearranjos cromossômicos recorrentes e não recorrentes. Este trabalho compreendeu o estudo de duas translocações cromossômicas aparentemente equilibradas e uma duplicação do braço curto do cromossomo 20 em decorrência de mosaicismo materno. O objetivo foi determinar os pontos de quebra por hibridação in situ fluorescente (FISH) e identificar genes candidatos, alterados pelas quebras dos rearranjos e que pudessem explicar o quadro clínico dos portadores. A caracterização das seqüências nos pontos de quebra e a junção desses rearranjos é fundamental para a compreensão dos mecanismos de formação das alterações cromossômicas. A delimitação precisa dos segmentos deletados é necessária para a correlação com o quadro clínico. / Two apparently \"de novo\" balanced translocations and one duplication of the short arm of chromosome 20 were studied. Our aim was to determine the breakpoints by chromosomal analysis through fluorescentin situ hybridization (FISH) and identify candidate genes and how they were involved with the clinical phenotypes of the patients. Patient 1 carried a duplication of the short arm of chromosome 20 (p11.22p13), inherited from the mother that showed normal and dup(20) lymphocytes. The duplication was determined by FISH using BAC and PAC clones, and nine clones were duplicated except one (20p11.21). The patient shared many of the common characteristics of trisomy 20p including delay in motor development, hypertelorism, poor coordination, round face with prominent cheeks, vertebral and dental abnormalities and cranial asymmetry with high and large forehead. She also had learning difficulties, behavioral disorders and pubertal growth spurt at 12 years. As our patient is an example of pure trisomy 20p, the features are of particular importance to delineate the syndrome. Three genes were mapped on the segment that contain the duplication (20p11.2-13), one of these genes is the SSTR4 (Somatostatin receptor 4). The somatostatin is widely distributed throughout the body and is important regulator of endocrine and nervous system function. It is an inhibitor of growth hormone secretion. The second gene is the BMP2 that produce bone morphogenetic proteins and it has a direct function with the nervous system. The third gene is the GHRH that produce proteins connected with the growth hormone. These genes might have been over expressed and thus contributing to the patient\'s clinical features. Patient 2, carried a 46,XY,t(5;14)(q14.1;q31.3)de novo translocation. On chromosome 14 the breakpoint was mapped to a segment contained in BAC RP11-315O17 (14q31.3). On the chromosome 5 the breakpoint was mapped to a segment contained in BAC RP11-30D15 (5q14.1). Although the breakpoint, on the chromosome 14, has been mapped in 14q31.3, our patient shared many of the common characteristics of terminal 14q32 deletion: mental retardation, dolicocephaly, prominent ears, hypertelorism, strabismus, upturned palpebral fissures, highly arched palate, simian crease, severe myopia, coloboma and palpebral ptosis. As mental retardation and ocular abnormalities were the main patient\'s clinical features, we are suggesting that: 1) a region of segment 14q31.3 was deleted. 2) A gene inside this segment (14q31.3) could be responsible for ocular development and 3) a disrupted gene could interfere on the expression of other genes. On chromosome 5 eleven genes were localized and four of them are expressed in nervous system (AP3B1; SCAMP1; BHMT2 e CMYA5). One of these genes might have been disrupted and is contributing to the patient\'s clinical features. Patient 3 was the carrier of a 46,XY,t(1;15)(p13.2;q25.2)de novo translocation. The breakpoint on chromosome 15 was mapped to the segment contained in clone RP11-152F13 (15q25.2). The breakpoint on chromosome 1 was mapped to the segment contained in clone RP5-1037B23 (1p13.2). The genes mapped at the breakpoint regions of chromosome 1 and chromosome 15 are expressed in nervous system and muscles. Our patient shows few clinical features: speech delay, stutter and learning difficulties, probably because one or more of these genes, mapped at the breakpoint region, could be disrupted.

Thesis (Ph. D.)--University of Nevada, Reno, 1999. / Includes bibliographical references. Online version available on the World Wide Web.

As alterações cromossômicas estruturais associadas a fenótipos clínicos oferecem a oportunidade de identificação e localização de genes cujas mutações possam estar determinando essas patologias, tendo em vista a possibilidade de que esses genes podem ter sido alterados pelas quebras ou ter o número de cópias modificado. Um número cada vez maior de evidências aponta para a participação de certas seqüências do genoma na formação de rearranjos cromossômicos recorrentes e não recorrentes. Este trabalho compreendeu o estudo de duas translocações cromossômicas aparentemente equilibradas e uma duplicação do braço curto do cromossomo 20 em decorrência de mosaicismo materno. O objetivo foi determinar os pontos de quebra por hibridação in situ fluorescente (FISH) e identificar genes candidatos, alterados pelas quebras dos rearranjos e que pudessem explicar o quadro clínico dos portadores. A caracterização das seqüências nos pontos de quebra e a junção desses rearranjos é fundamental para a compreensão dos mecanismos de formação das alterações cromossômicas. A delimitação precisa dos segmentos deletados é necessária para a correlação com o quadro clínico. / Two apparently \"de novo\" balanced translocations and one duplication of the short arm of chromosome 20 were studied. Our aim was to determine the breakpoints by chromosomal analysis through fluorescentin situ hybridization (FISH) and identify candidate genes and how they were involved with the clinical phenotypes of the patients. Patient 1 carried a duplication of the short arm of chromosome 20 (p11.22p13), inherited from the mother that showed normal and dup(20) lymphocytes. The duplication was determined by FISH using BAC and PAC clones, and nine clones were duplicated except one (20p11.21). The patient shared many of the common characteristics of trisomy 20p including delay in motor development, hypertelorism, poor coordination, round face with prominent cheeks, vertebral and dental abnormalities and cranial asymmetry with high and large forehead. She also had learning difficulties, behavioral disorders and pubertal growth spurt at 12 years. As our patient is an example of pure trisomy 20p, the features are of particular importance to delineate the syndrome. Three genes were mapped on the segment that contain the duplication (20p11.2-13), one of these genes is the SSTR4 (Somatostatin receptor 4). The somatostatin is widely distributed throughout the body and is important regulator of endocrine and nervous system function. It is an inhibitor of growth hormone secretion. The second gene is the BMP2 that produce bone morphogenetic proteins and it has a direct function with the nervous system. The third gene is the GHRH that produce proteins connected with the growth hormone. These genes might have been over expressed and thus contributing to the patient\'s clinical features. Patient 2, carried a 46,XY,t(5;14)(q14.1;q31.3)de novo translocation. On chromosome 14 the breakpoint was mapped to a segment contained in BAC RP11-315O17 (14q31.3). On the chromosome 5 the breakpoint was mapped to a segment contained in BAC RP11-30D15 (5q14.1). Although the breakpoint, on the chromosome 14, has been mapped in 14q31.3, our patient shared many of the common characteristics of terminal 14q32 deletion: mental retardation, dolicocephaly, prominent ears, hypertelorism, strabismus, upturned palpebral fissures, highly arched palate, simian crease, severe myopia, coloboma and palpebral ptosis. As mental retardation and ocular abnormalities were the main patient\'s clinical features, we are suggesting that: 1) a region of segment 14q31.3 was deleted. 2) A gene inside this segment (14q31.3) could be responsible for ocular development and 3) a disrupted gene could interfere on the expression of other genes. On chromosome 5 eleven genes were localized and four of them are expressed in nervous system (AP3B1; SCAMP1; BHMT2 e CMYA5). One of these genes might have been disrupted and is contributing to the patient\'s clinical features. Patient 3 was the carrier of a 46,XY,t(1;15)(p13.2;q25.2)de novo translocation. The breakpoint on chromosome 15 was mapped to the segment contained in clone RP11-152F13 (15q25.2). The breakpoint on chromosome 1 was mapped to the segment contained in clone RP5-1037B23 (1p13.2). The genes mapped at the breakpoint regions of chromosome 1 and chromosome 15 are expressed in nervous system and muscles. Our patient shows few clinical features: speech delay, stutter and learning difficulties, probably because one or more of these genes, mapped at the breakpoint region, could be disrupted.

Much of our understanding of eukaryotic life has come from studying mitochondrial DNA, giving rise to leading hypotheses in evolution. To enable these studies, efficient algorithms are needed to interpret, analyze, and draw relevant conclusions from the available mitochondrial sequence data. The central theme of this work is to provide such algorithms for two biological problems in mitogenomes. The key element of both methods is the de Bruijn graph. Small sequence segments of length k, called k-mers, of the genomes represented in the graph form the vertices. Two vertices are connected if the suffix of length (k-1) of the first vertex is equal to the prefix of length (k-1) of the second vertex. The edges are thus specified by the (k+1)-mer consisting of the k-prefix of the first vertex and the last character of the second vertex. The first problem is the automated accurate annotation of genes in complete mitochondrial sequences. For this purpose, a new method, called DeGeCI, is presented. The method uses a large collection of mitogenomes whose sequence data is represented as an annotated de Bruijn graph. To annotate an input genome sequence, initially, a subgraph induced by all (k+1)-mers of the sequence is constructed. Unmapped parts of the sequence result in disconnected components in this subgraph, which are bridged in the next step. For this purpose, alternative trails with a high sequence similarity to the respective unmapped subsequences of the input genome are identified in the database graph and added to the subgraph. Using a clustering approach, DeGeCI aggregates annotations contained in the resulting subgraph to obtain gene predictions for the input sequence. The thesis also presents the follow-up version of DeGeCI, which offers additional features and, in contrast to DeGeCI, can be used via a web server front-end. Genome rearrangements, which change the arrangement of the genes in the genome, are particularly common in mitogenomes. The locations in the genomes where the gene order differs are called breakpoints. The second objective of this thesis is to localize these breakpoints in the nucleotide sequences of complete mitochondrial genomes, taking into account possible high substitution rates. A novel method, DeBBI, is presented to address this task. The method constructs a colored de Bruijn graph of the input sequences, where each color is associated with one of the sequences. This graph is searched for certain structures that can be associated with the breakpoint locations. These so-called breakpoint bulges are common paths that branch into two separate paths and rejoin again at another location. One of the branches is short and of a single color, while the other branch is long and color-alternating. Sequence dissimilarities distort these structures by introducing additional branches. To identify the bulges despite these distortions, DeBBI uses a heuristic algorithm.

As alterações cromossômicas estruturais associadas a fenótipos clínicos oferecem a oportunidade de identificação de genes cujas mutações possam estar determinando essas patologias, tendo em vista a possibilidade de que esses genes podem ter sido alterados pelas quebras ou ter o número de cópias modificado. Um número cada vez maior de evidências aponta para a participação de certas seqüências do genoma na formação de rearranjos cromossômicos recorrentes e não recorrentes. Neste trabalho, estudamos duas deleções cromossômicas detectadas em indivíduos com retardo mental associado a sinais clínicos. O objetivo foi determinar que mecanismos originaram esses rearranjos e como a perda ou quebra dos segmentos cromossômicos está relacionada com o fenótipo dos portadores. A caracterização das seqüências nos pontos de quebra e junção desses rearranjos é fundamental para a compreensão dos mecanismos de formação das alterações cromossômicas. A delimitação precisa dos segmentos deletados é necessária para a correlação com o quadro clínico. Para isso, este trabalho aliou o estudo cromossômico por hibridação in situ fluorescente (FISH) à análise do DNA. / Structural chromosomal alterations related to clinical phenotypes bring the opportunity to identify gene mutations determining the pathologies, because the causative genes may have been disrupted by the breaks or may have an altered number of copies. The delimitation of the segments involved in the chromosomal rearrangements is necessary for these genotype-phenotype correlations. The characterization of breakpoint and junction sequences in these chromosome alterations enables the identification of mechanisms originating them, and evidence has been produced pointing to the participation of particular genomic sequences in their formation. In this work, we studied two chromosomal deletions in patients with syndromic mental retardation, combining chromosomal analysis by fluorescent in situ hybridization (FISH) to DNA analysis. Our aim was to determine the mechanisms that originated these aberrations and how they were involved with the clinical phenotypes.

As alterações cromossômicas estruturais associadas a fenótipos clínicos oferecem a oportunidade de identificação de genes cujas mutações possam estar determinando essas patologias, tendo em vista a possibilidade de que esses genes podem ter sido alterados pelas quebras ou ter o número de cópias modificado. Um número cada vez maior de evidências aponta para a participação de certas seqüências do genoma na formação de rearranjos cromossômicos recorrentes e não recorrentes. Neste trabalho, estudamos duas deleções cromossômicas detectadas em indivíduos com retardo mental associado a sinais clínicos. O objetivo foi determinar que mecanismos originaram esses rearranjos e como a perda ou quebra dos segmentos cromossômicos está relacionada com o fenótipo dos portadores. A caracterização das seqüências nos pontos de quebra e junção desses rearranjos é fundamental para a compreensão dos mecanismos de formação das alterações cromossômicas. A delimitação precisa dos segmentos deletados é necessária para a correlação com o quadro clínico. Para isso, este trabalho aliou o estudo cromossômico por hibridação in situ fluorescente (FISH) à análise do DNA. / Structural chromosomal alterations related to clinical phenotypes bring the opportunity to identify gene mutations determining the pathologies, because the causative genes may have been disrupted by the breaks or may have an altered number of copies. The delimitation of the segments involved in the chromosomal rearrangements is necessary for these genotype-phenotype correlations. The characterization of breakpoint and junction sequences in these chromosome alterations enables the identification of mechanisms originating them, and evidence has been produced pointing to the participation of particular genomic sequences in their formation. In this work, we studied two chromosomal deletions in patients with syndromic mental retardation, combining chromosomal analysis by fluorescent in situ hybridization (FISH) to DNA analysis. Our aim was to determine the mechanisms that originated these aberrations and how they were involved with the clinical phenotypes.