Les bactéries du genre Flavobacterium sont retrouvées dans des types d’habitats très divers. Ce genre contient trois espèces ichtyopathogènes : columnare, branchiophilum et psychrophilum qui est responsable de pertes économiques importantes pour l’élevage des salmonidés. Un projet de séquençage et de comparaison des génomes de plusieurs flavobactéries pathogènes de poissons ainsi qu’isolées de différents environnements a été mis en place pour améliorer les connaissances sur ce genre. Les objectifs étaient l’identification des déterminants de virulence et la caractérisation de différents marqueurs moléculaires des traits phénotypiques associés à leur mode de vie. L’analyse des génomes de F. psychrophilum a permis de mettre en évidence une diversité des structures chromosomiques au sein de l’espèce et d’identifier des cibles moléculaires prometteuses pour le développement de tests de diagnostic ainsi que des cibles vaccinales potentielles. Le génome de F. branchiophilum a permis d’identifier des mécanismes moléculaires de virulence originaux. Les caractéristiques du génome de F. indicum révèlent un mode de vie environnemental : sa petite taille et ses faibles capacités de dégradation des bio-polymères suggèrent que F. indicum est adapté à une niche écologique restreinte. Ces nouvelles données ont permis de caractériser in silico des marqueurs moléculaires de caractères phénotypiques. En particulier, un groupe de gènes (dnd) rare et responsable d’une modification étonnante de la molécule d’ADN a été décrit pour la première fois chez les Flavobacteriaceae. Ce projet a permis d’enrichir les connaissances sur les bactéries du genre Flavobacterium et a contribué au développement d’outils pour la santé animale. / Flavobacterium species occur in a wide range of habitats. This genus includes three fish-pathogenic species, namely F. columnare, F. branchiophilum and F. psychrophilum. The latter is responsible for serious economic losses for salmonids farming in France and worlwide. A comparative genomics project including several fish-pathogenic flavobacteria as well as various environmental species has been set up in order to improve the knowledge on this poorly studied genus. Our aims were the identification of virulence determinants associated with pathogenicity and the characterization of various molecular elements reflecting phenotypes associated with their life-style. Analysis of the genomes of several F. psychrophilum isolates revealed the diversity of chromosomal structures within the species and identified in silico promising molecular targets for the development of diagnostic tests as well as potential vaccines targets. Analysis of the F. branchiophilum genome enabled to identify particular molecular virulence mechanisms. The features of the F. indicum genome reflected its environmental lifestyle : its small size and its limited bio-polymers degrading abilities suggested that F. indicum is adapted to a quite narrow ecological niche. These new data have allowed the in silico identification of many molecular elements reflecting phenotypic traits. In particular, a rare gene cluster (dnd) responsible for an unusual DNA structure modification was described for the first time within members of the family Flavobacteriaceae. This project enriched the knowledge on Flavobacterium species and contributed to the development of tools for animal health.

Many problems in the biological sciences reduce to questions of genetic evolution. Entire classes of medical pathology, such as malignant neoplasia or infectious disease, can be viewed in the light of Darwinian competition of genomes. With the benefit of today's maturing sequencing technologies we can observe and quantify genetic evolution with nucleotide resolution. This provides a molecular view of genetic material that has adapted, or is in the process of adapting, to its local selection pressures. A series of problems will be discussed in this thesis, all involving the mathematical modeling of genomic data derived from clonally evolving populations. We use a variety of computational approaches to characterize over-represented features in the data, with the underlying hypothesis that we may be detecting fitness-conferring features of the biology. In Part I we consider the cross-sectional sampling of human tumors via RNA-sequencing, and devise computational pipelines for detecting oncogenic gene fusions and oncovirus infections. Genomic translocation and oncovirus infection can each be a highly penetrant alteration in a tumor's evolutionary history, with famous examples of both populating the cancer biology literature. In order to exert a transforming influence over the host cell, gene fusions and viral genetic programs need to be expressed and thus can be detected via whole transcriptome sequencing of a malignant cell population. We describe our approaches to predicting oncogenic gene fusions (Chapter 2) and quantifying host-viral interactions (Chapter 3) in large panels of human tumor tissue. The alterations that we characterize prompt the larger question of how the genetics of tumors and viruses might vary in time, leading us to the study of serially sampled populations. In Part II we consider longitudinal sampling of a clonally evolving population. Phylogenetic trees are the standard representation of a clonal process, an evolutionary picture as old as Darwin's voyages on the Beagle. Chapter 4 first reviews phylogenetic inference and then introduces a certain phylogenetic tree space that forms the starting point of our work on the topic. Specifically, Chapter 4 describes the construction of our projective tree space along with an explicit implementation for visualizing point clouds of rescaled trees. The Chapter finishes by defining a method for stable dimensionality reduction of large phylogenies, which is useful for analyzing long genomic time series. In Chapter 5 we consider medically relevant instances of clonal evolution and the longitudinal genetic data sets to which they give rise. We analyze data from (i) the sequencing of cancers along their therapeutic course, (ii) the passaging of a xenografted tumor through a mouse model, and (iii) the seasonal surveillance of H3N2 influenza's hemagglutinin segment. A novel approach to predicting influenza vaccine effectiveness is demonstrated using statistics of point clouds in tree spaces. Our investigations into clonal processes may be extended beyond naturally occurring genomes. In Part III we focus on the directed clonal evolution of populations of synthetic RNAs in vitro. Analogous to the selection pressures exerted upon malignant cells or viral particles, these synthetic RNA genomes can be evolved against a desired fitness objective. We investigate fitness objectives related to reprogramming ribosomal translation. Chapter 6 identifies high fitness RNA pseudoknot geometries capable of inducing ribosomal frameshift, while Chapter 7 takes an unbiased approach to evolving sequence and structural elements that promote stop codon readthrough.

Single-molecule carbon nanotube-based field-effect transistors are promising all-electronic devices for probing interactions of various biological and chemical molecules at the single- molecule level. Such devices consist of point-functionalized carbon nanotubes which are charge sensitive in the vicinity of a generated defect on the nanotube sidewall. Of particular interest is the characterization of the kinetic rates and thermodynamics of DNA duplex formation through repeated association (hybridization) and dissociation (melting) events on timescales unmatched by conventional single-molecule methods. In this work, we study the kinetics and thermodynamics of DNA duplex formation with two types of single-walled nanotubes: CVD-grown and solution-processed. In both assessments, we are able to extract kinetic and thermodynamic parameters governing the hybridization and melting of DNA oligonucleotides. In the latter case, devices are spun onto a wafer surface from an organic suspension, revealing consistent electrical characteristics. Significant effort is made to expand this work to wafer-level, in an effort to make the fabrication manufacturable.

The first half of this dissertation demonstrates development of a novel method for DNA methylation profiling based on site specific conversion of cytosine in CpG sites catalyzed by DNA methyltransferases. DNA methylation, a chemical process by which DNA bases are modified by methyl groups, is one of the key epigenetic mechanisms used by cells to regulate gene expression. It predominantly occurs at the 5-position of cytosines in CpG sites and is essential in normal development. Aberrant methylation is associated with many diseases including cancer. Bisulfite Genomic Sequencing (BGS), the gold standard in DNA methylation profiling, works on the principle of converting unmethylated cytosines to uracils using sodium bisulfite under strong basic conditions that cause extensive DNA damage limiting its applications. This dissertation focuses on the research and development of a new method for single cell whole-genome DNA methylation profiling that will convert the unmethylated cytosines in CpG sites to thymine analogs with the aid of DNA methyltransferase and photo-irradiation. Previously we synthesized a model deoxycytidine containing an optimized allyl chemical group at the 5-position and demonstrated that this molecule undergoes photo-conversion to its deoxythymidine analog (C to T conversion) with irradiation at 300 nm. The C to T conversion also proved feasible using synthetic DNA molecules. In this thesis, we demonstrate the conversion of a novel modified deoxycytidine molecule (PhAll-dC) using 350 nm photo-irradiation and a triplet photosensitizer (thioxanthone, TX) to avoid potential DNA damage. The new photoproduct was identified as the deoxythymidine analog of the starting molecule as assessed by IR, MS and NMR. An AdoMet analog containing the optimized chemical group was also synthesized and tested for enzymatic transfer to the C5-position of CpG cytosines using DNA methyltransferases. DNA methyltansferase M.SssI was engineered for more efficient enzymatic transfer. In the future, we will incorporate a triplet photosensitizer into the photoreactive moiety on AdoMet to increase energy transfer efficiency for photo-conversion of C to the T analog. Incorporating this into an overall method followed by amplification and sequencing should allow us to assess the methylation status of all CpGs in the genome in an efficient manner. The second half of this dissertation demonstrates development of a DNA sequencing by synthesis (SBS) method, The Sequence Walking Approach, using novel nucleotide reversible terminators (NRTs) together with natural nucleotides. Following the completion of The Human Genome Project, next generation DNA sequencing technologies emerged to overcome the limitations of Sanger Sequencing, the prominent DNA sequencing technology of the time. These technologies led to significant improvements in throughput, accuracy and economics of DNA sequencing. Today, fluorescence-based sequencing by synthesis methods dominate the high-throughput sequencing market. One of the major challenges facing fluorescence-based SBS methods is their read length limitation which constitutes a big barrier for applications such as de novo genome assembly and resolving structurally complex regions of the genome. In this regard, we have developed a novel SBS method called ‘The Sequence Walking Approach’ to overcome current challenges in increasing the single pass read length of DNA sequencing. Our method utilizes three dNTPs together with one nucleotide reversible terminator in reactions called ‘walks’ that terminate at predetermined bases instead of after each incorporation. In this method, the primer extended via 4-color SBS is stripped off and replaced by the original primer for walking reactions. By reducing the accumulation of cleavage artifacts of incorporated NRTs in a single run, our method aims to reach longer read lengths. In this thesis, we have demonstrated a variation of The Sequence Walking Approach in which 4-color sequencing steps are interspersed with walking steps over a continuous length of DNA without stripping off extended primers and reannealing the original primer. The improvements introduced with this method will enable the use of fluorescence-based SBS in many applications such as detection of genomic variants and de novo genome assemblies while preserving low costs and high accuracy.

Inspired by the TCGA (The Cancer Genome Atlas), we explore multimodal genomic datasets with integrative methods using a joint latent variable approach. We use iCluster+, an existing clustering method for integrative data, to identify potential subtypes within TCGA sarcoma and mesothelioma tumors, and across a large cohort of 33 dierent TCGA cancer datasets. For classication, motivated to improve the prediction of platinum resistance in high grade serous ovarian cancer (HGSOC) treatment, we propose novel integrative methods, iClassify to perform classication using a joint latent variable model. iClassify provides eective data integration and classication while handling heterogeneous data types, while providing a natural framework to incorporate covariate risk factors and examine genomic driver by covariate risk factor interaction. Feature selection is performed through a thresholding parameter that combines both latent variable and feature coecients. We demonstrate increased accuracy in classication over methods that assume homogeneous data type, such as linear discriminant analysis and penalized logistic regression, and improved feature selection. We apply iClassify to a TCGA cohort of HGSOC patients with three types of genomic data and platinum response data. This methodology has broad applications beyond predicting treatment outcomes and disease progression in cancer, including predicting prognosis and diagnosis in other diseases with major public health implications.

<p>Plant biomass is an abundant source of renewable energy, but the efficiency of its conversion into liquid fuels is low. One reason for this inefficiency is the recalcitrance of biomass to extraction and saccharification of cell wall polysaccharides. This recalcitrance is due to the complex and rigid structure of the plant cell wall. A better understanding of the genes effecting cell wall composition in bioenergy crops could improve feedstock quality and increase conversion efficiency. To identify genetic loci associated with biomass quality traits, we utilized genome-wide association studies (GWAS) in an 840-line <i>Sorghum</i> diversity panel. We identified several QTL from these GWAS including some for lignin composition and saccharification. Linkage disequilibrium (LD) analysis suggested that multiple polymorphisms are driving the association of SNPs within these QTL. Sequencing and further analysis led to the identification of a SNP within the coding region of a gene encoding phenylalanine ammonia-lyase (PAL) that creates a premature stop codon and co-segregates with an increase in the ratio of syringyl (S) to guaiacyl (G) lignin. A comparison of net PAL activity between lines with and without the mutation revealed that this mutation results in decreased PAL activity. </p>

In this thesis, we propose to discover co-regulated genes using microarray expression data, as well as providing visualization functionalities for domain experts to study relationships among discovered co-regulated genes. To discover co-regulated genes, we first use existing gene selection methods to select a small portion of genes which are relevant to the target diseases, on which we build an ordered similarity matrix by using nearest neighbor based similarity assessment criteria. We then apply a threshold based clustering algorithm named Spectral Clustering to the matrix to obtain a number of clusters. The genes which are clustered together in one cluster represent a group of co-regulated genes and to visualize them, we use Java Swings as the tool and develop a visualization platform which provides functionalities for domain experts to study relationships between different groups of co-regulated genes; study internal structures within each group of genes, and investigate details of each individual gene and of course for gene function prediction. Results are analyzed based on microarray expression datasets collected from brain tumor, lung cancers and leukemia samples. / by Vaibhan Lad. / Thesis (M.S.C.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

This aim of this research was to elucidate the genotypic diversity of the mycotoxigenic species Fusarium proliferatum associated with diverse hosts and distributed in wide geographic locations to gain new insights into the biology of this emerging pathogen. This study developed a novel molecular genetic marker FG1056. Multilocus typing of F. proliferatum isolates (52) using F. verticillioides (2) and F. oxysporum (3) as references was carried out with FG1056 and a set of known genetic markers (ITS, TEF1, CAL and FUM1). This distinguished up to 10 genetic groups, 2 clusters and 23 haplotypes among the F. proliferatum isolates. FG1056 marker showed the highest number of SNPs (169), informative sites (89) and haplotypes (23) relative to other markers used and was comparable to the multi locus typing. Varying patterns of relationships were observed between isolates represented in the genetic groups and their host and geographic origin. Considerable biological variability was recorded among the F. proliferatum isolates in morphology, growth, sporulation and most notably fumonisin production (up to 140-fold differences) with reference to variable temperature, water activity and duration. De novo genome assemblies with the size ranging from 43.96 - 50 Mb have been developed for four diverse F. proliferatum isolates. In silico analysis led to the identification of 12,980 genes common to all isolates and up to 134 genes potentially unique to an isolate. Using these resources, FUM gene cluster (~45.3 Kb) was identified for the first time in F. proliferatum. Order and orientation of the 16 FUM genes and the complete flanking genes (MSF1 and ZCB1 at 5’; ANK1 and GAT1 at 3’) have been determined. This study has provided new insights into the genetic and biological diversity of F. proliferatum and also developed new genetic and genomic resources, which will serve as a solid platform for further research particularly to understand the regulation of fumonisins production in the laboratory and in the field.

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No. of bitstreams: 1 paschoalotte_lm_dr_arafcl.pdf: 3219474 bytes, checksum: 3d1561985da9b8d968b144a96442458a (MD5) Previous issue date: 2018-04-03 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Há pelo menos três décadas a esfera pública vem sendo banhada pela figuração do humano como um ser de natureza igual – nem mais nem menos – a todos os outros seres viventes sob a rubrica da biologia molecular, mais precisamente da genômica. Do DNA como representação da “essência do nosso ser” aos “homens geneticamente criminosos”, vemos inúmeros enunciados serem vocalizados em livros, em reportagens e mídias em geral – especializados ou não – que, como diria Gyorgy Lukács, derivam ontologicamente as características do ser social daquelas constitutivas do ser natural. Desde a inauguração, na década de 1970 com a sociobiologia de Edward Wilson e Richard Dawkins, até os dias de hoje, a figuração do humano baseado na Teoria Sintética da Evolução vem se aperfeiçoando e se propagando nas distintas áreas do saber e da cultura. De forma geral, parte dominante desse pensamento interpreta as qualidades ontológicas dos humanos e, por consequência, suas características como resultados adaptacionista da evolução da nossa espécie com base na fitness genética. Sendo assim, no sentido de contribuir na compreensão do cenário no qual subiu ao palco tal figuração, este trabalho assume a tarefa de capturar alguns de seus significados históricosociais contemporâneos. Por consistir numa figuração com suportes teórico-científicos, a intenção, num primeiro momento, é identificar alguns dos seus fundamentos epistemológicos e ontológicos através da construção do que denominamos de grade de inteligibilidade genômico derivacionista, cuja característica central consiste na “dedução ontológica” das esferas menos complexas do ser em geral as mais complexas. Posteriormente, para levarmos a cabo o nosso objetivo, explicaremos o que consideramos efetivamente novo em seu significado histórico-social mediante as suas manifestações ideológicas – pelas quais práticas políticas e econômicas se operacionalizam. A nossa tese é de que, sob a crise estrutura do capital e seus aportes financeiros, emergiram tanto uma bioeconomia quanto uma biopolítica que imprimiram significados radicalmente novos ao modo com que tal figuração do humano se transmuta de discurso científico ao ideológico. / For at least three decades the public sphere has been bathed by the figuration of the human as a being of an equal nature – no more and no less – to all other living beings under the rubric of molecular biology, more precisely genomics. From DNA as a representation of the “essence of our being” to "genetically criminal men," we see innumerable utterances being spoken of in books, in reports, in advertisements and media in general – specialized or not – which, as Gyorgy Lukacs would say, derive ontologically the characteristics of the social being of those constitutive of the natural being. Since the inauguration in the 1970s with the sociobiology of Edward Wilson and Richard Dawkins, to this day, the human figure based on the Synthetic Theory of Evolution has been improving and spreading in the different areas of knowledge and culture. In general, a dominant part of this thought interprets the ontological qualities of humans and, consequently, their characteristics as an adaptational result of the evolution of our species based on genetic fitness. Thus, in order to contribute to the understanding of the scenario in which such figuration came to the stage, this work assumes the task of capturing some of its contemporary social-historical meanings. In the first place, the intention is to identify some of its epistemological and ontological foundations through the construction of what we call a “reductionist genomic intelligibility grid”, whose central characteristic consists of the "ontological deduction" of the less complex spheres of “being in general” the more complex. Subsequently, to accomplish our goal, we will explain what we consider to be effectively new in its historical-social meaning through its ideological manifestations – by which political and economic practices become operational. Our thesis is that, under the crisis of capital structure and its financial devices, both a bioeconomy and a biopolitics have emerged that have given radically new meanings to the way in which such figuration of the human transmutes from scientific to ideological discourse / 2014/27003-2

Bacteria have been the causative agents of major infectious disease pandemics throughout human history. Over the past 4 decades, a combination of changing medical practices, industrialization, and globalisation have led to a number of emergences and re-emergences of bacterial pathogens. The design of rational control programs and bespoke therapies will require an enhanced understanding of the dynamics underpinning the emergence and transmission of pathogenic clones. The recent development of new technologies for sequencing bacterial genomes rapidly and economically has led to a greatly enhanced understanding of the diversity of bacterial populations. This thesis describes the application of whole genome sequencing of 2 bacterial pathogens, Staphylococcus aureus and Legionella pneumophila, in order to understand the dynamics of bacterial infections on different temporal and spatial scales. The first study involves the examination of S. aureus evolution during a chronic infection of a single patient over a period of 26 months revealing differences in antibiotic resistance profiles and virulence factor expression over time. The genetic variation identified correlated with differences in growth rate, haemolytic activity, and antibiotic sensitivity, implying a profound effect on the ecology of S. aureus. Importantly, polymorphisms were identified in global regulators of virulence, with a high frequency of polymorphisms within the SigB locus identified, suggesting this region may be under selection in this patient. The identification of genes under diversifying selection during long-term infection may inform the design of novel therapeutics for the control of refractory chronic infections. Secondly, the emergence and transmission of 3 pandemic lineages derived from S. aureus clonal complex 30 (CC30) were investigated. Independent origins for each pandemic lineage were identified, with striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements, such as bacteriophage and Staphylococcal pathogenicity islands, and non-synonymous mutations affecting antibiotic resistance and virulence. Hospitals in large cities were identified as hubs for the transmission of MRSA to regional health care centres. In addition, comparison of whole genome sequences revealed that at least 3 independent acquisitions of TSST-1 have occurred in CC30, but a single distinct clade of diverse community-associated CC30 strains was responsible for the TSS epidemic of the late 1970s, and for subsequent cases of TSS in the UK and USA. Finally, whole genome sequencing was used as a tool for investigating a recent outbreak of legionellosis in Edinburgh. An unexpectedly high level of genomic diversity was identified among the outbreak strains, with respect to core genome polymorphisms, and accessory genome content. The data indicate that affected individuals may be infected with heterogeneous strains. The findings highlight the complexities in identifying environmental sources and suggest possible differences in pathogenic potential among isolates from a single outbreak. Taken together, the findings demonstrate applications of bacterial genome sequencing leading to enhanced understanding of bacterial pathogen evolution, emergence, and transmission, which may ultimately inform appropriate infection control measures.