One of the greatest challenges in modern biology is to understand quantitatively the mechanisms underlying messenger Ribonucleic acid (mRNA) transcription within the cell. To this end, integrated functional genomics attempts to use the vast wealth of data produced by modern large scale genomic projects to understand how the genome is deployed to create a diversity of tissues and species. The expression levels of tens or hundreds of thousands genes are profiled at multiple time points or different experimental conditions in the genomic projects. The profiling results are deposited in large scale quantitative data files that are not possible to analyse without systematic computational methods. In particular, it is much more difficult to experimentally measure the concentration level of transcription factor proteins and their affinity for the promoter region of genes, while it is relatively easy to measure the result of transcription using experimental techniques such as microarrays. In the absence of such biological experiments, it becomes necessary to use in silico techniques to determine the transcription factor regulatory activities given existing gene expression profile data. It therefore presents significant challenges and opportunities to the computer science community. This PhD Project made use of one such in silico technique to determine the differences (if any) in transcription factor regulatory activities of different experimental conditions and time points.The research aim of the Project was to understand the transcriptional regulatory mechanism that controls the sophisticated process of gene expression in cells. In particular, differences in the downstream signalling from which transcription factors can play a role in predisposition to diseases such as Parasitic disease, Cancer, and Neuroendocrine disease. To address this question I have had access to large integrated genomics datasets generated in studies on parasitic disease, lung cancer, and endocrine (hormone) disease. The current state-of-the-art takes existing knowledge and asks "How do these data relate to what we already know?" By applying machine learning approaches the project explored the role that such data can play in uncovering new biological knowledge.

Mammalian nuclear function depends on the complex interaction of genetic and epi-genetic elements coordinated in space and time. Structure and function overlap to such a degree that they are usually considered as being inextricably linked. In this work I combine an experimental approach with a computational one in order to answer two main questions in the field of mammalian chromosome organization. In the first section of this thesis, I attempted to answer the question, to what extent does chromatin from different chromosome territories share the same space inside the nucleus? This is a relatively open question in the field of chromosome territories. It is well-known and accepted that interphase chromosomes are spatially constrained inside the nucleus and that they occupy their own territory, however, the degree of spatial interaction between neighbouring chromosomes is still under debate. Using labelling methods that directly incorporate halogenated DNA precursors into newly replicated DNA without the need for immuno-detection or in situ hybridization, we show that neighbouring chromosome territories colocalise at very low levels. We also found that the native structure of DNA foci is partially responsible for constraining the interaction of chromosome territories as disruption of the innate architecture of DNA foci by treatment with TSA resulted in increased colocalisation signal between adjacent chromosomes territories. The second major question I attempted to answer concerned the correlation between nuclear function and the banding pattern observed in human mitotic chromosomes. Human mitotic chromosomes display characteristic patterns of light and dark bands when visualized under the light microscope using specific chemical dyes such as Giemsa. Despite the long standing use of the Giemsa banding pattern in human genetics for identifying chromosome abnormalities and mapping genes, little is known about the molecular mechanisms that generate the Giemsa banding pattern or its biological relevance. The recent availability of many genetic and epigenetic features mapped to the human genome permit a high-resolution investigation of the molecular correlates of Giemsa banding. Here I investigate the relationship of more than 50 genomic and epigenomic features with light (R) and dark (G) bands. My results confirm many classical results, such as the low gene density of the most darkly staining G bands and their late replication time, using genome-wide data. Surprisingly, I found that for virtually all features investigated, R bands show intermediate properties between the lightest and darkest G bands, suggesting that many R bands contain G-like sequences within them. To identify R bands that show properties of G bands, I employed an unsupervised learning approach to classify R bands on their genomic and epigenomic properties and show that the smallest R bands show a tendency to have characteristics typical of G bands. I revisit the evidence supporting the boundaries of G and R bands in the current cytogenomic map and conclude that inaccurate placement of weakly supported band boundaries can explain the intermediate pattern of R bands. Finally, I propose an approach based on aggregating data from multiple genomic and epigenomic features to improve the positioning of band boundaries in the human cytogenomic map. My results suggest that contiguous domains showing a high degree of uniformity in the ratio of heterochromatin and euchromatin sub-domains define the Giemsa banding pattern in human chromosomes.

A Bothrops jararaca é a serpente de maior importância médica no Brasil. Vários estudos foram realizados com o objetivo de caracterizar os componentes do veneno de serpentes, entretanto, a base molecular dos genomas das serpentes é pouco conhecida. Assim, foi realizado o sequenciamento e montagem do genoma da serpente Bothrops jararaca. Foram construídas bibliotecas tipo shotgun e mate-pair para realização de corridas de sequenciamento usando a tecnologia Illumina e sequências complementares foram obtidas em equipamento PACBIO RS II. Uma biblioteca de BACs também foi construída e 768 pools de 12 BACs foram sequenciados. Um grande conjunto de segmentos genômicos foi obtido e foi possível identificar genes de várias toxinas, entre elas SVMPs, SVSPs, BPPs, CRISPs e VEGF. Ainda foi possível depreender o contexto genômico de muitos destes genes e identificamos os principais elementos repetitivos genômicos. Estes achados são relevantes para o entendimento da função e evolução do sistema venenífero e podem servir de base para outros estudos futuramente. / The pit viper Bothrops jararaca is the most medically important snake in Brazil. Several studies were conducted in order to characterize the components of snake venom. However, the molecular basis of snake genomes is poorly known. Hence, it was carried out the sequencing and assembly of the Bothrops jararaca snake genome. Shotgun and mate-pair libraries were constructed to perform sequencing runs using Illumina technology and complementary sequences were obtained in PACBIO RS II equipment. A BAC library was also constructed and 768 pools of 12 BACs were sequenced. A large number of genomic segments was obtained. It was possible to identify genes of several toxins, including SVMPs, SVSPs, BPPs, CRISPs and VEGF. In addition, it was possible to infer the genomic context related to most of these genes and identify the main genomic repetitive elements. These findings are relevant for understanding the function and evolution of the venom system and it provides the basis for further studies.

Os elementos transponíveis (TEs) constituem grande parte do material genético de diversos eucariotos, alcançando entre 50-80% do genoma de gramíneas. Os projetos genoma proporcionaram um aumento das informações disponíveis sobre estes elementos, o que evidenciou sua importância e possibilitou o desenvolvimento de novas abordagens para seu estudo. O sistema Mutator (Mu) de milho é o mais ativo e mutagênico transposon de plantas. Além do elemento autônomo, MuDR, o sistema compreende ainda um conjunto de elementos bastante heterogêneo em sua seqüência e estrutura, chamados MuLEs, que podem conter até mesmo fragmentos de genes do hospedeiro. As seqüências de transposons mais abundantemente expressas no transcriptoma de cana-de-açúcar são relacionadas a MuDR e se agrupam em quatro clados (nomeados Classes I, II, II e IV), existentes antes da divergência entre Mono e Eudicotiledôneas. O trabalho apresentado aqui teve o objetivo de aprofundar o conhecimento sobre o sistema Mutator em cana-de-açúcar a partir da análise comparativa entre seqüências dessa planta e de arroz (cujo genoma está totalmente seqüenciado). Foi possível avaliar a abundância e diversidade do sistema Mu em gramíneas, ficando evidente uma amplificação de elementos clado-específica, tendo a Classe II sofrido uma explosão no número de cópias ao longo da evolução destas plantas. Análises estruturais revelaram que, enquanto as Classes I e II compreendem elementos com características de transposons, as Classes III e IV são, na verdade, transposases domesticadas. Foram completamente seqüenciados dois clones de BAC de cana-de-açúcar, um proveniente de cada parental do híbrido (Saccharum officinarum e Saccharum spontaneum), ambos contendo elementos da Classe III. Estes elementos foram caracterizados e a seqüência genômica de cana foi comparada com sua ortóloga em arroz, revelando um acúmulo de TEs nas regiões intergênicas. / Transposable elements (TEs) constitute great part of eukaryote genetic material, in grasses, they comprise between 50-80% of the genome. Genome projects have significantly increased the amount of information about these elements, revealing their importance and allowing the development of new approaches for their study. The Mutator system (Mu) of maize is the most active and mutagenic plant transposon. Beyond the autonomous element, MuDR, the system comprises a very heterogeneous, in sequence and structure, set of elements, called MuLEs, that can contain even host gene fragments. The most abundant transposon related sequences expressed in sugarcane transcriptoma are the MuDR-like. They group into four clades (called Classes I, II, III and IV) that exist prior to the Mono and Eudicot split. The aim of this work is to gain knowledge about the Mutator system in sugarcane through the comparative analysis against rice (whose genome is completely sequenced). The results described the abundance and diversity of the Mu system in grasses, evidencing a clado-specific amplification with a burst of Class II along the evolution of this plant group. Structural analyses showed that, while Classes I and II comprise elements with transposon characteristics, Classes III and IV are domesticated transposases. One BAC clone from each sugarcane parental genotype (Saccharum officinarum and Saccharum spontaneum) have been completely sequenced, both containing Class III elements. These elements have been characterized and the sugarcane genomic sequences were compared with their orthologues in rice. The comparative analyses showed an accumulation of TEs in the intergenic regions.

No description available.

Since the inception of the Human Genome Project (HGP) there has, and continues to be, rapid changes in genomics, STEM, and human health. Advances, specifically in genomics, continue to be increasingly important as new knowledge in this field has led the trajectory for significant advancements in all biological disciplines. Throughout the scientific community there is an emphasis on increasing and improving genomic concepts and literacy for grades K-12. Numerous research studies report that there is generally a low level of genetic/genomic knowledge among the general public. The purpose of this research is to analyze and document evidence of secondary school students’ participation, and educational outcomes, in a novel course on genomic principles and practices. A mixed methods approach, using qualitative and quantitative methods was used to address three research questions. 1) Based on affective evidence, how did secondary school students perceive and critically judge, content topics learned in a course on modern genomic principles and practices? 2) Based on cognitive evidence, how much of the content did secondary school students learn when they participated in a course on modern genomic principles and practices? 3) Using individual interview evidence, what are the major perceptions that the secondary school students expressed throughout the duration of the course? The results for Research Question 1 demonstrated that the students gained a significant level of new knowledge pertaining to genomics after attending the course sessions, based on their pre-and post-test Likert survey data. More particularly, they expressed more interest in, and understanding of genomic principles and practices. Concurrently, they became much more critically reflective and evaluative about some of the societal and medical implications of its applications. With respect to Research Question 2, the secondary school students’ content knowledge as measured by a 25-question multiple-choice pre-and-post test administered before and after the course demonstrated a significant increase. Lastly, the participants were provided an opportunity to comment on the course through individual and collaborative interviews, in order to find out to what extent they perceived the course to be interesting and challenging. Future inquiry expanding from this research would help to establish the foundational pathway for designing a more inclusive genomics curriculum.

O etanol possui grande importância estratégica para o setor energético brasileiro devido a sua natureza renovável e grande capacidade de geração de emprego e renda. Atualmente um dos principais desafios enfrentados pelas indústrias brasileiras de produção de etanol é a presença de bactérias contaminando as fermentações. Essas atuam modificando o sistema, competindo por nutrientes essenciais, produzindo compostos tóxicos para a levedura e induzindo processos indesejáveis para a indústria como a floculação. Para equilibrar as populações e controlar os efeitos negativos é necessário uma melhor compreensão dos mecanismos genéticos e fisiológicos envolvidos nas interações de bactérias e leveduras em fermentações em fermentações etanólicas de primeira geração (1G). Neste sentido o presente trabalho realizou o sequenciamento, montagem e anotação completa dos genomas de três linhagens bacterianas isoladas diretamente de usinas brasileiras de produção de etanol: Lactobacillus sp. I-2, Lactobacillus sp. L-10 e Acetobacter sp. A-3. Além disso, foram feitas simulações da condição de co-existência entre bactérias lácticas e Acéticas com as leveduras de S. cerevisiae em fermentações semi-industriais em batelada alimentada (\"Melle-Boinot\") com tratamento ácido e reciclo de células. Três condições experimentais foram avaliadas: (i) fermentações contendo apenas leveduras de S. cerevisiae CAT-1, (ii) fermentações contendo leveduras de S. cerevisiae CAT-1 e bactérias de Lactobacillus sp. I-2, e (iii) fermentações contendo leveduras de S. cerevisiae CAT-1 e bactérias de Acetobacter sp. A-3. Por fim, os metabolomas das bactérias e leveduras da condição de coexistência nos ensaios fermentativos foram obtidos por metodologias de cromatografia gasosa acoplada a espectrometria de massas (GC-MS). Foram identificados 3056, 2112 e 3557 genes nos genomas de Lactobacillus sp. L-10, Lactobacillus sp. I-2 e Acetobacter sp. A-3, respectivamente. As leveduras de S. cerevisiae, independente da condição experimental avaliada, consumiram totalmente os açúcares redutores totais (ART) presentes no melaço de cana-de-açúcar ao longo das fermentações em batelada alimentada. A glicina, aminoácido polar contendo apenas um hidrogênio na cadeia lateral, apresentou maior abundância durante as fermentações contaminadas com bactérias lácticas e acéticas em detrimento da condição controle sem contaminação bacteriana. Especula-se que o acúmulo intracelular deste aminoácido seja resultado do desvio de intermediários das vias Glicólise e Ciclo do Ácido Cítrico para a produção de compostos nitrogenados. Provavelmente, ambos os microrganismos, bactérias e leveduras, acumularam glicina no ambiente intracelular como alternativa a deficiência de nitrogênio no melaço de cana-de-açúcar. O sequenciamento dos genomas das bactérias e os ensaios fermentativos em biorreatores foram realizados no Laboratório Nacional de Biorrenováveis (LNBR) do Centro Nacional de Pesquisas em Energias e Materiais (CNPEM). As análises de metabolômica foram realizadas no Laboratório Max Feffer de Genética de Plantas da Escola Superior de Agricultura \"Luiz de Queiroz\" (ESALQ). Espera-se que os resultados apresentados neste trabalho agreguem mais informações a cerca das interações entre bactérias e leveduras e contribua, num futuro próximo, no apontamento de novas estratégias de controle bacteriano que sejam mais eficientes e sustentáveis que as usadas atualmente em fermentações industriais brasileiras. / Ethanol has great strategic importance for the Brazilian energy sector due to its renewable nature and great capacity to generate jobs and income. Currently, one of the main challenges faced by the Brazilian ethanol industry is the presence of contaminating bacteria in the fermentation tanks. They act by modifying the system, competing for essential nutrients, producing toxic compounds to the yeast and inducing undesirable processes for the industry like flocculation. To better balance populations and to control the negative effects, it is necessary a better understanding of the genetic and physiological mechanisms involved in the interactions among bacteria and yeasts from Saccharomyces cerevisiae. In this sense, the present work carried out the sequencing, assembly and complete genome annotation of three bacterial strains isolated directly from Brazilian ethanol production plants: Lactobacillus sp. I-2, Lactobacillus sp. L-10 and Acetobacter sp. A-3. In addition, simulations of the co-existence condition between lactic and acetic bacteria with yeasts from S. cerevisiae were carried out in semi-industrial fed-batch (Melle-Boinot) fermentations with acid treatment and cell recycling. Three experimental conditions were evaluated: (i) fermentations containing only yeasts from S. cerevisiae CAT-1, (ii) fermentations containing yeasts of S. cerevisiae CAT-1 and bacteria from Lactobacillus sp. I-2, and (iii) fermentations containing yeasts from S. cerevisiae CAT-1 and bacteria from Acetobacter sp. A-3. Finally, the metabolomes of bacteria and yeasts in the coexistence condition on fermentation assays were obtained by gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled to mass spectrometry (LC-MS). 3056, 2112 and 3557 genes were identified in the genomes of Lactobacillus sp. I-2, Lactobacillus sp. L-10 and Acetobacter sp. A-3, respectively. The yeasts from S. cerevisiae, regardless of the experimental condition evaluated, all the total reducing sugars (ART) present in sugarcane molasses were consumed and the ethanol production was consistent with that observed in first generation (1G) ethanolic fermentations. Glycine, an apolar amino acid containing only one hydrogen in the lateral chain, showed greater abundance during fermentations contaminated with lactic and acetic bacteria. It is speculated that the intracellular accumulation of this amino acid results from the shift of intermediates from the Glycolysis/Glycogenogenesis and the Citric Acid Cycle pathways to the production of nitrogen compounds. Probably, both microorganisms, bacteria and yeasts, used the intracellular accumulation of glycine as an alternative to nitrogen deficiency in sugarcane molasses in the fermentations carried out in bioreactors. The Bacterial genomes sequencing and fermentation assays in bioreactors were performed at the Brazilian Bioethanol Science and Technology Laboratory (CTBE) of the Brazilian Center for Research in Energy and Materials (CNPEM). The metabolomics analyzes were performed at the Max Feffer Laboratory of Plant Genetics at the \"Luiz de Queiroz\" School of Agriculture (ESALQ). It is hoped that the results presented in this work will add more information about the interactions among bacteria and yeasts and will contribute, in the near future, to new bacterial control strategies in Brazilian industrial fermentations that are more efficient and sustainable than those currently used.

Pas de résumé / No abstract

The human gut microbiome is a vast ecosystem of microorganisms that play an important role in human metabolism, immunological function, and even inflammatory gut diseases. Metagenomics research on the human gut microbiome has demonstrated the presence of DNA from dietary yeast species like Saccharomyces cerevisiae. However, it is unknown if the S. cerevisiae detected in metagenomics studies is solely from dead dietary sources or if they can live and colonize the human gut like their close relative Candida albicans. While S. cerevisiae can adapt to low oxygen and acidic environments, it has yet to be explored whether it can metabolize mucin, the primary carbon source found in the mucus layer of the human gut. Mucins are large, gel-forming, highly glycosylated proteins that make up a majority of carbohydrate sources in the gut mucosa. This work determined that S. cerevisiae can utilize mucin as their main carbon source which results in a significant reduction in cell size. Additionally, an aspartyl protease named Yps7, part of a family containing known homologues to mucin-degrading C. albicans proteins in S. cerevisiae, is important for growth on mucin media. To further identify biological pathways required to grow optimally in mucin, both a transcriptome analysis on wild type cells (BY4743) and a chemogenomics screen was performed. In total, 2131 genes demonstrated significant differential expression in mucin media, and 30 genes upon their deletion impacted their growth on mucin. Both these screens suggest that mitochondrial function is required for proper growth in mucin, which was further elucidated by the change in mitochondrial morphology and oxygen consumption in yeast cells upon mucin treatment. Indeed, the uncharacterized open reading frame YCR095W-A is required for growth on mucin as the deletion mutant showed dysfunction in mitochondrial morphology and cellular respiration, further suggesting a potential role in mitochondrial function. Importantly, this project serves as the initial step towards establishing if our most common dietary fungus can survive in the mucus environment of the human gut.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22354号 / 医博第4595号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 武藤 学, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM