Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Brachyspira hyodysenteriae and Brachyspira pilosicoli are anaerobic intestinal spirochaetes that are the aetiological agents of swine dysentery and intestinal spirochaetosis, respectively. As part of this PhD study the genome sequence of B. hyodysenteriae strain WA1 and a near complete sequence of B. pilosicoli strain 95/1000 were obtained, and subjected to comparative genomic analysis. The B. hyodysenteriae genome consisted of a circular 3.0 Mb chromosome, and a 35,940 bp circular plasmid that has not previously been described. The incomplete genome of B. pilosicoli contained 4 scaffolds. There were 2,652 and 2,297 predicted ORFs in the B. hyodysenteriae and B. pilosicoli strains, respectively. Of the predicted ORFs, more had similarities to proteins of the enteric Clostridium species than they did to proteins of other spirochaetes. Many of these genes were associated with transport and metabolism, and they may have been gradually acquired through horizontal gene transfer in the environment of the large intestine. A reconstruction of central metabolic pathways of the Brachyspira species identified a complete set of coding sequences for glycolysis, gluconeogenesis, a non-oxidative pentose phosphate pathway, nucleotide metabolism and a respiratory electron transport chain. A notable finding was the presence of rfb genes on the B. hyodysenteriae plasmid, and their apparent absence from B. pilosicoli. As these genes are involved in rhamnose biosynthesis it is likely that the composition of the B. hyodysenteriae lipooligosaccharide O-sugars is different from that of B. pilosicoli. O-antigen differences in these related species could be associated with differences in their specific niches, and/or with their disease specificity. Overall, comparison of B. hyodysenteriae and B. pilosicoli protein content and analysis of their central metabolic pathways showed that they have diverged markedly from other spirochaetes in the process of adapting to their habitat in the large intestine. The presence of overlapping genes in the two spirochaetes and in other spirochaete species also was investigated. The number of overlapping genes in the 12 spirochaete genomes examined ranged from 11-45%. Of these, 80% were unidirectional. Overlapping genes were found irregularly distributed within the Brachyspira genomes such that 70-80% of them occurred on the same strand (unidirectional, ->->/<-<-), with 16-28% occurring on opposite DNA strands (divergent, <-->). The remaining 4-6% of overlapping genes were convergent (-><-). The majority of the unidirectional overlap regions were relatively short, with >50% of the total observations overlapping by >4 bp. A small number of overlapping gene-pairs were duplicated within each genome and there were some triplet overlapping genes. Unique orthologous overlapping genes were identified within the various spirochaete genera. Over 75% of the overlapping genes in the Brachyspira species were in the same or related metabolic pathway. This finding suggests that overlapping genes are not only likely to be the result of functional constraints but also are constrained from a metabolomic context. Of the remaining 25% overlapping genes, 50% contained one hypothetical gene with unknown function. In addition, in one of the orthologous overlapping genes in the Brachyspira species, a promoter was shared, indicating the presence of a novel class of overlapping gene operon in these intestinal spirochaetes.

Thesis--University of Oklahoma. / Includes bibliographical references.

Thesis--University of Oklahoma. / Includes bibliographical references.

La diversité d’une espèce est représentée par la somme de la diversité de chacun des individus qui la compose. Elle peut être observée à différentes échelles : individuelle, organique, tissulaire, cellulaire, génomique, génique, ou bien à l’échelle de la base nucléotidique. L’étude de la diversité d’une espèce est importante pour mieux la comprendre et nous permettre de retracer son histoire évolutive, de la comparer avec d’autres espèces notamment entre espèces sauvages et cultivées. Nous nous intéressons aux processus de domestication, et particulièrement à leurs impacts sur la structure du pan-génome. Le pan-génome est divisé en trois compartiments : (i) le core-génome qui contient tous les gènes présents chez tous les individus de l’espèce ; (ii) le génome dispensable qui contient l’ensemble des gènes qui sont absents chez au moins un individu ; (iii) et enfin le génome individu-spécifique qui contient les gènes présents uniquement chez un individu.L’objectif de ce travail de thèse était de mettre au point une nouvelle méthode d’analyse pan-génomique applicable sur un grand nombre d’individus. Pour cela, nous avons travaillé sur un jeu de données de reséquençage massif du riz Africain cultivé O. glaberrima et de son ancêtre sauvage O. barthii. Dans un premier temps nous avons vérifié l’existence d’une structure pan-génomique sur notre modèle. Pour cela nous avons travaillé à petite échelle avec trois accessions de l’espèce cultivée. Elles ont d’abord été séquencées, assemblées, annotées puis nous avons cherché à détecter des séquences spécifiques à chacune de ces accessions.Dans un second temps nous avons mis au point notre méthode en travaillant avec près de 200 génomes des deux espèces.Ces génomes ont été séquencés grâce aux technologies NGS puis directement mappés sur un génome de référence externe, celui du riz Asiatique. Nous avons alors appliqué notre méthode d’analyse pan-génomique basée sur la déviation de la profondeur deséquençage pour chaque gène. Nous avons ensuite comparé les enrichissement d’ontologies par compartiments et par espèce dans le but d’identifier des différences liées aux processus de domestication. Enfin, nous avons étudié plus précisément les appartenances pan-génomiques des membres de famille de gènes.Parce que le pan-génome de l’espèce cultivé est plus petit que le core-génome de l’espèce sauvage nous avons confirmé la perte de diversité en terme de présence/ absence de gènes chez le riz Africain au cours du processus de domestication. Curieusement nous avons aussi mis en avant l’augmentation du nombre de gènes dispensable chez l’espèce cultivée par rapport à son relatif sauvage.Ainsi, malgré une forte réduction du pan-génome de l’espèce cultivé lors de la « première » sélection, les 1000 générations de processus de domestication ont suffit à réintroduire une forme de diversité à travers l’augmentation du nombre de gènes dispensables.Afin d’automatiser une grande partie des manipulations d’analyses de données NGS nous avons aussi développé un outil de génération de pipelines d’analyses. De part sa généricité et sa robustesse il pourra être utilisé dans différents domaines, pour plu-sieurs types de données. Grâce aux nombreux logiciels qui y sont intégrés et de par le suivi que l’équipe de développement entend poursuivre, il pourra être utilisé dans la caractérisation de plus en plus de choses. Par exemple les variations structurales, les associations génotypes-phénotypes, l’épigénétique et pourquoi pas la métagénomique.Ce travail a permis la mise au point d’une nouvelle méthode d’analyse des données pan-génomiques rapide de par sa vision globale plutôt que via des comparaisons deux-à-deux. Cette méthode s’adresse aux génomes grands et complexes comme ceux des plantes, mais aussi aux jeux de données massifs. / Species diversity is represented by the sum of the diversity of each of the individuals composing it. It can be seen at differents cales: individual, organic, tissular, cellular, genomic, gene, and even nucleotic. The study of the diversity of species is important to better understand and allow tracking its evolutionary history, comparing it to other species, in particular wild to cultivated. We focused on the domestication, and particularly its impact on the pan-genome structure.The pan-genome is divided into three compartments: (i) the core-genome containing all the genes present in all individuals of the species; (ii) the dispensable genome containing all genes absent in at least one individual; (iii) and finally the individual-specific genome containing genes present only in one individual.The objective of this thesis was to develop a new method for pan-genomic analysis that can apply to a large number of indi-viduals. For this, we worked on a massive resequencing data set of cultivated African rice O. glaberrima and of its wild ancestor O. barthii. At first we checked the existence of a pan-genomic structure on our model. For this we worked on a small scale, with three accessions of cultivated species. They were sequenced, assembled, annotated then analyzed to detect specific sequences for each accession.Secondly we developed our approach working with nearly 200 genomes of both species. These genomes were sequenced using Illumina technology and mapped to the external reference genome, of the Asian rice. We applied our pan-genomic method analysis based on the deviation of the depth of sequencing for each gene. We then compared the ontology enrichment compartments and species in order to identify differences related to the domestication process. Finally, we looked specifically to pan-genomic genes belonging to gene family. Because the pan-genome of the cultivated species is smaller than the core-genome of the wild one, we confirmed the loss ofdiversity in terms of presence/ absence of genes in African rice during the domestication process. Curiously we have also high lighted the increase in the number of dispensable genes in the crop from its wild relative. Thus, despite a sharp reduction of the pan-genomeof the species cultivated in the “first” selection, the 1,000 generations of domestication process were enough to reintroduce a formof diversity through increasing the number of dispensable genes.To automate much of the data analysis of NGS manipulations we have also developed a tool to generate analysis pipelines.Due to its generic and robustness it can be used in different areas, for several types of data. With many softwares integrated and by monitoring that the development team will continue, it may be used in the characterization of more and more things. For example,structural variations, genotype-phenotype associations, epigenetics and metagenomics. This work enabled the development of a new analytical method for rapid genome-wide data through its global vision ratherthan through two by two comparisons. This method is for large and complex genomes such as those of plants, but also to massivedata sets.

O presente estudo teve como objetivo caracterizar o genoma de estirpes de Leptospira isoladas no Brasil e realizar a análise comparativa destes com os genomas disponíveis no banco de dados GenBank. Foram caracterizadas 17 estirpes isoladas de distintas espécies animais, em diferentes regiões do Brasil, no período de 1998 a 2012. Estas foram previamente tipificadas por sequenciamento do gene 16S rRNA e soroaglutinção microscópica em seis espécies (L. interrogans, L. santarosai, L. inadai, L. kirschneri, L. borgpetersenii e L. noguchii) e mais de oito sorogrupos. Foi realizado o sequenciamento em plataforma Illumina&trade; MiSeq e montagem dos genomas com algoritmo ab initio. Para ordenação e anotação foram utilizados genomas de referência das respectivas espécies estudadas. Foi realizada a análise in silico da Tipagem por Sequenciamento de Multilocus (MLST) para os três protocolos vigentes de Leptospira. A análise comparativa dos genomas, incluindo wgSNP, foi realizada intra-espécie avaliando as variações existentes entre os sorogrupos das espécies de Leptospira estudadas. As estirpes de L. interrogans apresentaram resultados na MLST congruentes com a sua identificação prévia. No caso de L. kirschneri, apenas uma estirpe apresentou novos alelos nos três protocolos de MLST e se distancia das demais estirpes brasileiras de L. kirschneri. As estirpes de L. santarosai, assim como as de L. borgpetersenii e L. noguchii, possuem novos alelos e/ou perfis alélicos para pelo menos dois dos protocolos vigentes de MLST, sendo que ainda se destacam em um agrupamento próprio de origem brasileira. Os genomas de L. interrogans apesar de apresentarem alta identidade e sintenia com a referência sorovar Copenhageni, também apresentaram regiões de diferença entre os respectivos sorogrupos. Os genomas dos sorogrupos Australis e Serjoe se destacaram por apresentarem inserções e deleções, respectivamente, principalmente no cromossomo 2. O genoma de L. borgpetersenii também apresentou grande variação de composição, como esperado para espécie, sendo esta proporcionada por sequências de inserção e transposição de elementos móveis. Os sorogrupos Canicola e Pomona apresentaram maior proximidade entre si na análise wgSNP. Também foram identificados dois plasmídeos nos genomas do sorogrupo Canicola com alta identidade aos plasmídeos descritos na estirpe chinesa do mesmo sorovar. Na espécie L. kirschneri, a estirpe 47 (M36/05) apresentou alta identidade e sintenia com os genomas do sorovar Mozdok, como esperado, incluindo a estirpe brasileira de origem humana. Já a estirpe 55 (M110/06) se diferenciou dos demais genomas de L. kirschneri tanto no MLST quanto no wgSNP. O genoma brasileiro de L. inadai apresentou alta identidade à referência americana de origem humana, incluindo a presença de um bacteriófago próprio da espécie. A distinção das estirpes brasileiras de L. santarosai na MLST, também foi evidenciada na análise comparativa e no wgSNP, sendo que a estirpe 68 (M52/8-19), que não apresentou reatividade aos sorogrupos testados, ainda se diferencia das demais reafirmando a possibilidade de novo sorogrupo/sorovar. Dessa forma, o estudo genômico possibilitou a identificação de particularidades das estirpes brasileiras de Leptospira, incluindo a existência de elementos extra-cromossomais, proximidade com estirpes de origem humana indicando maior risco para saúde pública, além da possibilidade de novo sorogrupo de L. santarosai. / The present study aimed to characterize the genome of Leptospira strains isolated in Brazil and to perform their comparative analysis with GenBank available genomes. 17 strains isolated from distinct species, in different regions of Brazil, from 1998 to 2012 were characterized. These were previously typified through 16S rRNA sequencing and microscopic agglutination into six species (L. interrogans, L. santarosai, L. inadai, L. kirschneri, L. borgpetersenii and L. noguchii) and over eight serogroups. Illumina&trade; MiSeq sequencing and genome assembly with ab initio algorithm were performed. For ordering and annotation, reference genomes of the respective species were used. The in silico analysis of Multilocus Sequencing Typing (MLST) was performed for the three current Leptospira protocols. The comparative genomic analysis, including wgSNP, was performed intra-species evaluating the existing variations between the serogroups of the studied Leptospira species. The L. interrogans strains presented MLST results congruent with their previous identification. In the case of L. kirschneri, only one strain presented new alleles in the three MLST protocols and distanced itself from the other Brazilian L. kirschneri strains. The L. santarosai strains, as well as L. borgpetersenii and L. noguchii, presented new alleles and/or allelic profiles for at least two of the current MLST protocols, and still stand out in a separate group of Brazilian origin. Even though the L. interrogans genomes presented high identity and synteny with serovar Copenhageni reference, they also presented regions of difference between the respective serogroups. Serogroups Australis and Serjoe genomes stood out for having insertions and deletions, respectively, mainly in chromosome 2. The L. borgpetersenii genome also presented great variation of composition, as expected for the species, which is provided by insertion sequences and transposition of mobile elements. The serogroups Canicola and Pomona presented higher proximity in the wgSNP analysis. Two plasmids were also identified in the serogroup Canicola genomes with high identity to the plasmids described in the Chinese strain of the same serovar. In the L. kirschneri species, the strain 47 (M36/05) presented high identity and synteny with the serovar Mozdok genomes, as expected, including the Brazilian strain of human origin. The strain 55 (M110/06) differed from other L. kirschneri genomes in both MLST and wgSNP. The Brazilian L. inadai genome presented high identity to the American reference of human origin including the presence of bacteriophage specific for the species. The distinction of the Brazilian L. santarosai strains in the MLST was also evidenced in the comparative analysis and in the wgSNP, and the strain 68 (M52 / 8-19), which showed no reactivity to the tested serogroups, also differs from the others reaffirming the possibility of a new serogroup/serovar. Therefore, the genomic study allowed the identification of particularities of Brazilian Leptospira strains, including the existence of extrachromosomal elements, proximity to strains of human origin indicating a greater risk for public health, in addition to the possibility of a new L. santarosai serogroup.

A endometriose é uma doença ginecológica benigna comum, mas agressiva, caracterizada pela presença de tecido endometrial ectópico. A teoria mais aceita para explicá-la é a teoria de Sampson, na qual o tecido endometrial descamado durante a menstruação sofre refluxo através das tubas uterinas, adere e se prolifera em sítios ectópicos da cavidade peritoneal. Por outro lado, apenas o refluxo tubário não é capaz de estabelecer a doença e vários estudos sugerem uma etiologia multidimensional incluindo fatores hereditários, hormonais e imunológicos. Várias metodologias têm sido propostas com o objetivo de identificar genes candidatos para a endometriose. A hibridação genômica comparativa (CGH) é uma técnica que permite que o genoma inteiro seja analisado em um só experimento, sem a necessidade de cromossomos metafásicos obtidos por cultura celular. Nossa proposta foi avaliar, por CGH, amostras de endometriomas ovarianos e de tecido endometrial eutópico de dez pacientes com diagnóstico firmado de endometriose, para screening do genoma. No grupo eutópico, 6/10 amostras apresentaram alterações caracterizadas por perdas ou ganhos de regiões cromossômicas e no grupo ectópico foram encontradas alterações em 7/10 casos. A presença de perdas e ganhos de regiões cromossômicas no endométrio eutópico, histologicamente normal, de mulheres com endometriose ovariana, pode ser considerada como alteração primária ao desenvolvimento da doença. A metodologia de CGH permitiu a detecção das regiões cromossômicas 11q12.3-q13.1, 17p11.1-p12 e 17q25.3-qter como regiões críticas, direcionando investigações futuras para identificação de genes associados à endometriose. / Endometriosis is a common benign gynecological disease, very aggressive, characterized by the presence of ectopic endometrial tissue. The most accepted theory to explain it is Sampson\'s implantation theory, which says that the endometrial tissue exfoliated during menstruation undergoes reflux through the uterine tubes, adheres and proliferates in ectopic sites of the peritoneal cavity. On the other hand, only reflux is not enough to the establishment of the disease and a number of studies suggest a multidimensional etiology including hereditary, hormonal and immunological factors. Several methodologies have been proposed for the identification of candidate genes for endometriosis. The comparative genomic hybridization (CGH) is a versatile technique that allows the entire genome to be analyzed in only one experiment without the necessity of metaphasic chromosomes from the sample, excluding the cell culture. We aimed to evaluate, by CGH, ovarian endometriomas and eutopic endometrial tissue samples from 10 patients with confirmed diagnosis of endometriosis, for a genomic screening. In the eutopic group, 6/10 samples presented genomic imbalances and 7/10 cases showed alterations in the ectopic group. The presence of losses and gains of chromosomic regions in the histologically normal eutopic endometrium from women with ovarian endometriosis can be considered as a primary alteration in the development of the disease. The CGH methodology allowed the detection of chromosomic regions 11q12.3-q13.1, 17p11.1-p12 and 17q25.3-qter as critical regions, leading to future investigations for the identification of genes associated to endometriosis.

Orientadores: Luciana Bolsoni Lourenço, Carmen Silvia Busin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T12:08:20Z (GMT). No. of bitstreams: 1 Gatto_KalebPretto_M.pdf: 33988822 bytes, checksum: 7e3f3565dac9a540ea7c353f51068d4f (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

No description available.