As pesquisas em biologia molecular têm produzido uma grande quantidade de dados, os quais embutem informações sobre diferentes fenômenos biológicos. Neste sentido, a bioinformática se destaca como uma área de pesquisa multidisciplinar que visa, principalmente, o desenvolvimento de ferramentas (sistemas) computacionais para auxiliar na descoberta de conhecimento a partir de dados biológicos. Dentro da bioinformática, a área de genômica funcional procura estudar as funções gênicas através da medição simultânea e em larga escala dos níveis de expressão gênica de um genoma. Diferentes ferramentas são utilizadas no processo de análise de expressão gênica, cada qual provê suporte a uma atividade de análise específica. Embora alguns ambientes de descoberta de conhecimento ofereçam suporte integrado a este processo de análise e exploração de dados, a maior parte das ferramentas de análise é desenvolvida independentemente de outras ferramentas e ambientes de descoberta de conhecimento. Este cenário representa um desafio para biologistas que precisam combinar e integrar diferentes ferramentas, muitas vezes de forma ad hoc, custosa e sujeita a erros. Modelos conceituais, tais como ontologias, têm contribuído para o sucesso do desenvolvimento de sistemas computacionais em diferentes domínios de aplicação. O desenvolvimento de tais modelos tem por objetivo representar corretamente, em alto nível de abstração, conceitos e situações pertinentes a um dado domínio de interesse. Esta representação abstrata facilita não apenas o entendimento de um dado domínio, mas também serve como base para o processo de desenvolvimento do sistema como um todo. O objetivo deste trabalho é investigar o desenvolvimento e o uso de modelos conceituais em geral e ontologias em particular, na integração de ferramentas na área de análise de expressão gênica. De forma específica, este trabalho tem por objetivo propor uma abordagem para a integração semântica de ferramentas de análise de expressão gênica a partir do uso de conectores e de uma ontologia de domínio. Essa abordagem foi aplicada no desenvolvimento de estudos de caso envolvendo a criação de diferentes ambientes integrados para a análise de expressão gênica e mostrou-se eficaz. / Molecular biology researches are increasingly producing large amounts of data regarding underlying biological phenomena. Bioinformatics is a multidisciplinary research field whose main objective is the development of theories and information systems to help the process of knowledge discovery from biological data. Functional genomics is a field of study bioinformatics concerned with the study of gene function through parallel and large scale expression measurements of a genome. A variety of software tools are usually combined and used in a knowledge discovery process, each providing support for a specific data analysis task. Although some tools are already provided as part of an integrated knowledge discovery environment, most of them are developed independently of other software tools and knowledge discovery environments. This scenario poses a problem and a challenge for biologists that need to combine and integrate different tools in an ad hoc, time consuming and error prone process. Conceptual models, such as ontologies, have contributed to the successful development of information systems in different application domains. The development of such models aims at creating a clear and precise description of the elements of a given domain at a high abstraction level. This abstract and high level description not only promotes a shared understanding of the domain, but also serves as basis for the development process of supporting applications in the domain. This work aims at investigating the development and use of conceptual models in general and ontologies in particular to support the integration of gene expression data analysis systems. Specifically, this work proposes an approach for the semantic integration of gene expression analysis tools using connectors and a domain ontology. This approach was applied in the development of a number of case studies aiming at creating integrated environments for gene expression analysis and proved its effectiveness.

Made available in DSpace on 2014-08-20T13:42:10Z (GMT). No. of bitstreams: 1 Dissertacao_Joceani_Dal_Cero.pdf: 1522635 bytes, checksum: 6a5a31f6f6667ef5f524442ba0bb1e72 (MD5) Previous issue date: 2010-02-26 / Regulatory molecules, such as transcription factors, have been thoroughly investigated, especially in hormone-mediated responses that involve gene expression modulation. Frequently, the main determinant of gene expression is its transcriptional rate. Thus, molecular mechanisms underlying transcription regulation have become an important topic in genetic studies of ethylene signaling. The present work aimed to investigate the ERF (Ethylene Response Factor) family employing bioinformatic tools, integrating publicly available datasets from the model species Arabidopsis thaliana and phylogenetic analyses to help elucidating the biological roles of the family in apple. The preliminary survey of the ERF sequences in Malus has provided basic information to be incorporated in further studies of the functional role of ERFs in this perennial species. Expression analyses of MdERF1 and MdERF in apple fruits suggest that other factors, besides ethylene, are involved in their transcriptional regulation in Malus. The second chapter reports the investigation of the transcriptional profiling of those ERF genes in response to pathogen attack, using a biological assay of in vitro propagated plants inoculated with the fungus Venturia inaequalis (apple scab disease). The study has provided evidences of the involvement of MdERF1 in eliciting the plant response; whereas, MdERF2 does not appear to be participate in the pathogenesis. / Moléculas que participam dos processos regulatórios, como os fatores de transcrição, têm recebido atenção especial, pois uma das principais ações dos estímulos hormonais é a modulação da expressão gênica. Como a taxa de transcrição de um gene é o maior determinante da sua expressão, os mecanismos moleculares pelos quais a transcrição gênica é regulada têm se tornado um dos tópicos principais de estudos em genética molecular envolvendo o hormônio etileno. O objetivo deste trabalho foi realizar análises de bioinformática para a família ERF (Ethylene Response Factors), integrar bases de dados existentes na internet no modelo Arabidopsis, bem como análise filogenética que permitam avaliar os papéis dos diferentes membros da família. Este levantamento preliminar das seqüências ERF em Malus forneceu informação básica para estudos posteriores mais aprofundados, com relação aos mecanismos moleculares da família nesta importante cultura perene. A análise da expressão de MdERF1 e MdERF2 em frutos de maçã indica que outros fatores além do etileno estão envolvidos na regulação da transcrição dos ERF em Malus. O segundo capítulo refere-se à resposta dos ERF frente ao ataque de patógenos. Para isso, foram infectadas plantas de macieira provenientes de cutivo in vitro com o fungo Venturia inaequalis (sarna da maçã). As evidências desses estudos sugerem o envolvimento do gene MdERF1 no processo de patogênese, enquanto que o gene MdERF2 parece não estar envolvido no processo.

O fungo Aspergillus fumigatus é o segundo maior causador de infecções fúngicas invasivas em pacientes imunocomprometidos e a principal espécie causadora da aspergilose invasiva, doença de alta taxa de mortalidade que atinge principalmente os pulmões e que pode se disseminar pelo organismo. Durante o processo de infecção, o fungo precisa adaptar-se ao organismo do hospedeiro e um dos obstáculos encontrados é a mudança na concentração de dióxido de carbono (CO2), que, de 0,033% no ambiente, chega a até 6% no interior do hospedeiro. As anidrases carbônicas são enzimas envolvidas na hidratação reversível do CO2 e já foram apontadas como importantes na virulência de patógenos como Plasmodium falciparum, Mycobacterium tuberculosis, Helicobacter pylori, Cryptococcus neoformans e Candida albicans. Esse trabalho teve como objetivo avaliar o papel da enzima anidrase carbônica no desenvolvimento e virulência do fungo A. fumigatus, que apresenta quatro homólogos desta enzima (cafA, cafB, cafC e cafD). Para isso, foram utilizadas linhagens de A. fumigatus com os homólogos da enzima deletados (?cafA, ?cafB, ?cafC, ?cafD e ?cafA?cafB) e a linhagem selvagem (?akuBku80), da qual foram originadas as mutantes. Foram realizadas avaliações fenotípicas da estrutura dos conidióforos das diferentes linhagens, determinação da sensibilidade frente a diferentes agentes estressantes (antifúngicos, promotores de apoptose, estresse iônico, nitroativo, oxidativo, e de parede celular) e determinação da expressão gênica global em diferentes concentrações de CO2. Foi verificado que a deleção de cada um dos homólogos da anidrase carbônica de A. fumigatus não interfere na estrutura dos conidióforos deste fungo. Por outro lado, a deleção induziu alteração da sensibilidade do fungo frente a alguns compostos estressantes (ácido acético e peróxido de hidrogênio). Ainda, a análise da expressão gênica revelou um gene envolvido na adaptação do fungo ao aumento da concentração de CO2, o gene cipC, que não apresenta homólogos nas células de mamíferos. Este gene foi caracterizado neste trabalho por meio de sua deleção na linhagem selvagem (?akuBku80) de A. fumigatus e avaliação fenotípica microscópica e de sensibilidade a agentes estressantes (antifúngicos, promotores de apoptose, estresse iônico, nitroativo, oxidativo, e de parede celular). A deleção do gene não interferiu na estrutura do fungo, porém aumentou sua sensibilidade a alguns compostos (calcoflúor e menadiona). Foram realizados, ainda, testes de virulência em modelo animal utilizando-se o mutante ?cipC, os quais revelaram que a deleção deste gene atenua a virulência do fungo. Assim, foi possível concluir que as anidrases carbônicas não são relevantes para o desenvolvimento e virulência de A. fumigatus; porém, este fungo modifica a expressão de seus genes de modo a adaptar-se às variações na concentração atmosférica de CO2. O gene cipC está envolvido nesse processo de adaptação e é importante para o desenvolvimento do fungo e sua virulência, tornando-se um alvo para o estudo de novas terapias para o tratamento da aspergilose invasiva. / The fungus Aspergillus fumigatus is the second cause of fungal infections in immunocompromised patients and it is the main specie which causes invasive aspergillosis, a disease with high mortality rate that mainly affects the lungs and it can spread through the body. During the infectious process, the fungus must adapt to the host and one of the obstacles is the drastic change of the carbon dioxide (CO2) concentration, which is 0.033% in the environment and until 6% inside the host. The carbonic anhydrases are enzymes which are involved in the reversible hydration of carbon dioxide and they have been pointed as important in the virulence of pathogens such as Plasmodium falciparum, Mycobacterium tuberculosis, Helicobacter pylori, Cryptococcus neoformans and Candida albicans. This work aimed to evaluate the role of the enzyme carbonic anhydrase in the development and virulence of the fungus A. fumigatus, which has four homologues of this enzyme (cafA, cafB, cafC e cafD). Therefore, strains, which have the homologues of the enzyme deleted (?cafA, ?cafB, ?cafC, ?cafD and ?cafA?cafB) were used in parallel with the wild strain (?akuBku80), which originated the mutant ones. We did structure phenotypic evaluations of the different strains of conidiophores, sensibility determination against different stressors (antifungal agents, apoptosis, ionic, nitrosative, oxidative, and cell wall stress promoters) and global gene expression determination at different carbon dioxide concentrations. It was verified that the carbonic anhydrases homologues deletion of A. fumigatus did not interfere on the n structure (conidiophore) of this fungus, in the tested conditions. On the other hand, the deletion caused a change in sensibility of the fungus against some stressors (acetic acid and hydrogen peroxide). The gene expression experiments showed a gene involved in the adaptation to the increase of CO2 concentration, the cipC gene. This gene does not have homologues in the mammalian cells. The cipC gene was characterized in this work by its deletion in the A. fumigatus wild strain (?akuBku80) and microscopic phenotypic evaluation and sensibility tests against stressors (antifungal agents, apoptosis, ionic, nitrosative, oxidative, and cell wall stress promoters). The gene deletion did not interfere on the fungus conidiophore structure but increase its sensibility to some compounds (calcoflúor white and menadione). Virulence tests in animal model using the ?cipC mutant were done and they showed that the deletion of this gene attenuates the fungus virulence. In conclusion, the carbonic anhydrases are not relevant to development and virulence of the fungus, which modifies the gene expression to adapt to the variations of atmospheric CO2 concentration. Besides, the cipC gene seems to be involved in this adaptation process. Moreover, the cipC gene showed to be important to the development of the fungus and its virulence, which makes the gene a target for the study of new therapies for the treatment of invasive aspergillosis.

Estudos de expressão gênica geralmente envolvem a realização de processos de análise integrados para a obtenção de respostas biológicas de interesse. A realização destes processos frequentemente requer o uso combinado de uma série de ferramentas de software. No entanto, o processo de integração manual de ferramentas pode ser demorado e propenso a erros devido ao crescente número de ferramentas e formatos de dados disponíveis no domínio. De modo a automatizar o processo de integração, algumas abordagens têm sido propostas tanto para a adaptação das ferramentas de análise existentes como serviços web semânticos, quanto para o desenvolvimento de ambientes de suporte à integração (composição) de serviços web semânticos. Embora estas abordagens representem avanços, nenhuma solução adequada para o desenvolvimento e composição de serviços foi especificamente definida para o domínio de genômica funcional. Neste contexto, o principal objetivo deste projeto foi investigar uma solução completa para o desenvolvimento e composição de serviços web semânticos para a análise de expressão gênica. Como parte da solução proposta, definimos uma metodologia integrada para a implementação de serviços web semânticos criados a partir de ferramentas de software existentes e para a anotação semântica destes serviços. Nossa metodologia fornece diretrizes concretas para o desenvolvimento sistemático de serviços, considerando também os principais aspectos técnicos associados ao processo de desenvolvimento. Esta metodologia foi aplicada a um conjunto representativo de serviços que fornecem suporte às principais atividades de análise realizadas em diferentes tipos de dados de expressão gênica. De forma complementar, definimos uma solução completa para a composição semântica de serviços no domínio de análise de expressão gênica. A solução proposta foi implementada em uma plataforma de suporte semi-automático à composição de serviços web semânticos, chamada SemanticSCo. Esta plataforma fornece suporte flexível a todas as atividades envolvidas no processo de composição de serviços, incluindo a criação, publicação, requisição, descoberta, seleção, composição e execução de serviços. Além disto, a plataforma SemanticSCo foi projetada para prover suporte adequado a diferentes tipos de usuários, incluindo biologistas e bioinformatas. Neste sentido, a plataforma fornece aos usuários um alto nível de abstração para a definição de seus processos de análise, permitindo que os mesmos se concentrem mais nas questões de pesquisa biológicas do que nos aspectos subjacentes do processo de composição. Adicionalmente, a plataforma SemanticSCo suporta a definição e incorporação não apenas de serviços simples, definidos em termos de uma única operação, mas também de serviços complexos, definidos em termos de um conjunto de condições que restringem a ordem de invocação de suas operações. Finalmente, de modo a avaliar a plataforma de suporte desenvolvida, definimos diferentes cenários de composição para a análise (integrada) de dados de expressão gênica. O uso da plataforma SemanticSCo facilitou a definição destes cenários, permitindo assim a reprodução dos resultados obtidos a partir de diferentes estudos de expressão gênica previamente documentados na literatura / Gene expression studies usually involve the creation of integrated analysis processes for obtaining responses for a biological question. The creation of such processes often require the combined use of a number of software tools. However, the manual integration of tools can be cumbersome and error prone due to the increasing number of tools and data formats available in the domain. In order to automate the integration process, some approaches have been proposed for the adaptation of existing analysis tools as semantic web services as well as for the development of software environments to support the integration (composition) of semantic web services. Although these approaches present advances, to the best of our knowledge, no suitable solution has been proposed for the development and composition of web services in the functional genomics domain. In this context, this project aimed at investigating a complete solution for the development and composition of semantic web services to support gene expression analysis. As part of the proposed solution, we have defined an integrated methodology for the implementation of semantic web services created from existing software tools and the semantic annotation of such services. Our methodology provides concrete guidelines for the systematic development of services, also taking into account the main technical aspects associated with the development process. This methodology has been applied in the development of a representative set of services that support the main analysis activities performed on different types of gene expression data. Complementary to our methodology, we have defined a complete solution for the semantic composition of web services in the gene expression analysis domain. The proposed solution has been implemented in a software platform to support the semi-automatic composition of semantic web services, named SemanticSCo. This platform provides flexible support to all activities involved in the service composition process including service creation, publication, request, discovery, selection, composition and execution. Additionally, the SemanticSCo platform has been designed to support different types of users, including biologists and bioinformaticians. In this sense, the platform provides users with a high level of abstraction in the definition of their analysis processes, thus allowing them to focus more on biological research issues rather than on underlying details of the composition process. In addition, the SemanticSCo platform supports not only the definition and incorporation of (simple) services defined in terms of a single operation, but also (complex) services defined in terms of a set of conditions that constrain the order in which service operations should be invoked. Finally, in order to evaluate the developed support platform, we have defined a number of composition scenarios for the (integrated) analysis of gene expression data. The use of the SemanticSCo platform has facilitated the definition of these scenarios, thus allowing the reproduction of the results obtained from different gene expression studies previously documented in the literature.

En Amérique Latine, les punaises hématophages Triatominae transmettent à l’homme le parasite Trypanosoma cruzi, responsable de la maladie de Chagas touchant actuellement 5 millions de personnes. Même si les programmes d’éradication chimique des vecteurs sont efficaces, la maladie persiste du fait de la recolonisation des habitations humaines par des vecteurs provenant d’habitats naturels. Ainsi, certaines espèces présentent une capacité d’adaptation aux anthroposystèmes (processus de domiciliation), alors que d’autres espèces apparentées ne l’ont pas. Comprendre cette capacité d’adaptation est crucial d’un point de vue épidémiologique afin de cibler les espèces présentant un risque pour l’homme. La capacité à s’adapter à un nouvel habitat pourrait être liée à l’évolution du répertoire de gènes du système chimiosensoriel, important pour la perception du milieu. Cette étude a porté sur le système chimiosensoriel des Triatominae dans le but de documenter le processus d’adaptation et donc de domiciliation des vecteurs. Des données transcriptomiques obtenues en séquençage à haut débit ont été utilisées pour annoter et répertorier les gènes chimiosensoriels ainsi que pour comparer leur expression au sein de punaises hématophages d’habitats différents. L’existence d’une relation entre les variations de ces gènes chez différentes espèces de Triatominae et leur capacité d’adaptation à un habitat a par la suite été évaluée. L’espèce T. brasiliensis en voie de domiciliation au Brésil et présentant à la fois des populations sylvatiques, péri-domiciliaires et domiciliaires, et différentes espèces du genre Rhodnius d’habitats variés, ont été étudiées, notamment les deux espèces sœurs, R. robustus, sylvatique en Amazonie et R. prolixus majoritairement domiciliée dans toute son aire de répartition. En l’absence de génomes de références suffisamment proches de T. brasiliensis et des 10 espèces de Rhodnius étudiées, leurs transcriptomes ont été assemblés de novo. Les transcriptomes des deux espèces R. prolixus et R. robustus ont été assemblés par alignement sur le génome de R. prolixus. Chez ces différentes espèces de Triatominae étudiées, l’analyse du répertoire des gènes chimiosensoriels codant les OBPs et CSPs (familles multigéniques) comparé à celui d’autres Paranéoptères a montré des expansions géniques pouvant refléter des processus adaptatifs. Par ailleurs, chez les différentes espèces du genre Rhodnius, il existe une corrélation positive entre le nombre de gènes codant les OBPs et la capacité de domiciliation, suggérant l’implication de cette famille de gènes dans l’adaptation au milieu anthropique. Les analyses d’expression différentielle concernant les différentes populations de T. brasiliensis et les espèces R. prolixus/R. robustus ont montré qu’un certain nombre de transcrits sont différentiellement exprimés selon l’environnement dans lequel ont évolué les punaises notamment des gènes chimiosensoriels (OBPs, CSPs) ainsi que des gènes impliqués dans le rythme circadien et le comportement de recherche alimentaire (Takeout), dans la réponse à des stress environnementaux comme des gènes de détoxification (P450, glutathione S-transférase), dans la résistance à des changements climatiques (Heat-shock protéines) et dans la protection du milieu extérieur (protéines cuticulaires). Ce travail a permis de mettre à la disposition de la communauté scientifique des outils performants pour l’étude du processus de domiciliation des vecteurs de la maladie de Chagas (transcriptome, répertoire de gènes). Il a également permis de révéler des gènes qui pourraient être impliqués dans l’adaptation et/ou la plasticité phénotypique en réponse à un changement d’habitat. La compréhension des bases moléculaires de l’adaptation des vecteurs aux habitations humaines ouvre des potentialités de développer des méthodes alternatives de lutte contre les vecteurs qui pourraient être basées sur une perturbation de la communication chimique. / In Latin America, the bloodsucking bugs (Triatominae, Hemiptera, Reduviidae) are vectors of the parasite Trypanosoma cruzi, which causes Chagas disease. More than five million people are infected. Even if chemical control campaigns are effective against vectors, the disease persists due to the recolonization of human habitations by vectors from natural habitats. Some species have the capacity to adapt to anthroposystems (domiciliation process), while other related species do not. Understanding this capacity to adapt is crucial from an epidemiological perspective to target species at risk to humans. The capacity to adapt to a new habitat could be linked to changes in the repertoire of chemosensory system genes, particularly for odorant binding proteins (OBP) and chemosensory proteins (CSP), which are important proteins to detect various odor stimuli. This study is based on the chemosensory system of Triatominae to document the adaptation process and then the domiciliation of the vectors. Transcriptomic data obtained by high-throughput sequencing were used to annotate and list the chemosensory genes and also to compare their expression in bloodsucking bugs from different habitats. The relationship between changes in these genes in different Triatominae species and their ability to adapt to a new habitat was evaluated. The species T. brasiliensis, which is in the process of domiciliation in Brazil with sylvatic, peridomiciliary and domiciliary populations, and various species of the genus Rhodnius from diverse habitats were studied, especially the two sibling species R. robustus, sylvatic in the Amazonia and R. prolixus mostly domiciliary throughout its geographical range. In the absence of a reference genome for T. brasiliensis, a reference transcriptome via de novo assembly (data 454 and Illumina) was achieved. The reference transcriptomes for 10 Rhodnius species were also established using the de novo assembly method. A genome reference based method on R. prolixus was also used to assemble the transcriptome of the two species R. prolixus and R. robustus. In the different species of the Triatominae studied, the chemosensory gene repertoire showed a high diversity and genic expansions compared to that of others Paraneoptera, which could reflect adaptive process. Furthermore, a positive correlation was shown between the number of OBP genes in Rhodnius species and their domiciliation ability, suggesting that this gene family is involved in the adaptation to anthropogenic environment. The differential expression analyses on the T. brasiliensis populations and the R. prolixus / R. robustus species showed that some transcripts are differentially expressed according to the environment in which the bugs have evolved, especially the chemosensory genes (OBP, CSP) and also genes involved in the circadian rhythm and foraging behavior (Takeout), in the response to environmental stress such as detoxification genes (P450, glutathione S-transferase), in resistance to climatic changes (heat-shock proteins) and in protection from the external environment (cuticular proteins).This work has helped make available to the scientific community powerful tools for studying the process of domiciliation of Chagas disease vectors (transcriptome, gene repertoire). It also revealed genes that could be involved in the adaptation and/or phenotypic plasticity in response to a change in habitat. Understanding the molecular basis of vector adaptation to human dwellings opens the potential to develop new tools to control the disease vectors, for example by disrupting chemical communication.

Differential expression analysis can be a powerful method to investigate expressed differences between closely related species. Our ambition is to highlight differentially expressed nuclear genes to explain the hybrid incompatibilities among the Amazonian, Centro-American and Orinocan semispecies of Drosophila paulistorum. RNA sequencing (RNA-seq) establishes the foundation of the study where we first evaluate the influence of two distinct alignment references. We discover the benefits of concatenating a de novo assembly instead of using the genome reference of a close relative. The bioinformatic pipeline handles the interesting inclusion of D. melanogaster and D. willistoni, where their contribution assists in the search for previously studied speciation genes. Among the down- and upregulated subsets we can see a diverse mix of general biological processes such as regulatory functions and transcriptional factors. In the end we uncover potential indications to why the Amazonian seems to be the least compatible semispecie to produce hybrids. This study provides a competitive working frame for comparative RNA-seq studies between closely related species.

<p>For many years, researchers have developed ways to diagnose degenerative disease in the retina by utilizing multiple gene analysis techniques. Retinitis pigmentosa (RP) disease can cause either partially or totally blindness in adults. For that reason, it is crucial to find a way to pinpoint the causes in order to develop a proper medication or treatment. One of the common methods is genome-wide analysis (GWA). However, it cannot fully identify the genes that are indirectly related to the changes in eye size. In this research, RNA sequencing (RNA-seq) analysis is used to link the phenotype to genotype, creating a pool of candidate genes that might associate with the RP. This will support future research in finding a therapy or treatment to cure such disease in human adults.</p> <p><br></p> <p>Using the Drosophila Genetic Reference Panel (DGRP) – a gene reference panel of fruit fly – two types of datasets are involved in this analysis: eye-size data and gene expression data with two replicates for each strain. This allows us to create a phenotype-genotype map. In other words, we are trying to trace the genes (genotype) that exhibit the RP disease guided by comparing their eye size (phenotype). The basic idea of the algorithm is to discover the best replicate combination that maximizes the correlation between gene expression and eye-size. Since there are 2N possible replicate combinations, where N is the number of selected strains, the original implementation of sequential algorithm was computationally intensive.</p> <p><br></p> <p>The original idea of finding the best replicate combination was proposed by Nguyen et al. (2022). In this research, however, we restructured the algorithms to distribute the tasks of finding the best replicate combination and run them in parallel. The implementation was done using the R programming language, utilizing doParallel and foreach packages, and able to execute on a multicore machine. The program was tested on both a laptop and a server, and the experimental results showed an outstanding improvement in terms of the execution time. For instance, while using 32 processes, the results reported up to 95% reduction in execution time when compared with the sequential version of the code. Furthermore, with the increment of computational capabilities, we were able to explore and analyze more extreme eye-size lines using three eye-size datasets representing different phenotype models. This further improved the accuracy of the results where the top candidate genes from all cases showed connection to RP.</p>

Weed resistance to control measures, particularly herbicides, is a growing problem in agriculture. In the case of herbicides, resistance is sometimes connected to genetic changes that directly affect the target site of the herbicide. Other cases are less straightforward where resistance arises without such a clear-cut mechanism. Understanding the genetic and gene regulatory mechanisms that may lead to the rapid evolution of resistance in weedy species is critical to securing our food supply. To study this phenomenon, we exposed young Arabidopsis plants to sublethal levels of one of four weed management stressors, glyphosate herbicide, trifloxysulfuron herbicide, mechanical clipping, and shading. To evaluate responses to these stressors we collected data on gene expression and regulation via epigenetic modification (methylation) and small RNA (sRNA). For all of the treatments except shade, the stress was limited in duration, and the plants were allowed to recover until flowering, to identify changes that persist to reproduction. At flowering, DNA for methylation bisulfite sequencing, RNA, and sRNA were extracted from newly formed rosette leaf tissue. Analyzing the individual datasets revealed many differential responses when compared to the untreated control for gene expression, methylation, and sRNA expression. All three measures showed increases in differential abundance that were unique to each stressor, with very little overlap between stressors. Herbicide treatments tended to exhibit the largest number of significant differential responses, with glyphosate treatment most often associated with the greatest differences and contributing to overlap. To evaluate how large datasets from methylation, gene expression, and sRNA analyses could be connected and mined to link regulatory information with changes in gene expression, the information from each dataset and for each gene was united in a single large matrix and mined with classification algorithms. Although our models were able to differentiate patterns in a set of simulated data, the raw datasets were too noisy for the models to consistently identify differentially expressed genes. However, by focusing on responses at a local level, we identified several genes with differential expression, differential sRNA, and differential methylation. While further studies will be needed to determine whether these epigenetic changes truly influence gene expression at these sites, the changes detected at the treatment level could prime the plants for future incidents of stress, including herbicides. / Doctor of Philosophy / Growing resistance to herbicides, particularly glyphosate, is one of the many problems facing agriculture. The rapid rise of resistance across herbicide classes has caused some to wonder if there is a mechanism of adaptation that does not involve mutations. Epigenetics is the study of changes in the phenotype that cannot be attributed to changes in the genotype. Typically, studies revolve around two features of the chromosomes: cytosine methylation and histone modifications. The former can influence how proteins interact with DNA, and the latter can influence protein access to DNA. Both can affect each other in self-reinforcing loops. They can affect gene expression, and DNA methylation can be directed by small RNA (sRNA), which can also influence gene expression through other pathways. To study these processes and their role in abiotic stress response, we aimed to analyze sRNA, RNA, and DNA from Arabidopsis thaliana plants under stress. The stresses applied were sublethal doses of the herbicides, glyphosate and trifloxysulfuron, as well as mechanical clipping and shade to represent other weed management stressors. The focus of the project was to analyze these responses individually and together to find epigenetic responses to stresses routinely encountered by weeds. We tested RNA for gene expression changes under our stress conditions and identified many, including some pertaining to DNA methylation regulation. The herbicide treatments were associated with upregulated defense genes and downregulated growth genes. Shade treated plants had many downregulated defense and other stress response genes. We also detected differential methylation and sRNA responses when compared to the control plants. Changes to methylation and sRNA only accounted for about 20% of the variation in gene expression. While attempting to link the epigenetic process of methylation to gene expression, we connected all the data sets and developed computer programs to try to make correlations. While these methods worked on a simulated dataset, we did not detect broad patterns of changes to epigenetic pathways that correlated strongly with gene expression in our experiment's data. There are many factors that can influence gene expression that could create noise that would hinder the algorithms' abilities to detect differentially expressed genes. This does not, however, rule out the possibility of epigenetic influence on gene expression in local contexts. Through scoring the traits of individual genes, we found several that interest us for future studies.

No description available.

Aim: To develop an immunomagnetic assay for the isolation of circulating tumor cells (CTCs) followed by the analysis of a multimarker panel, which will enable the characterization of these malignant cells with high accuracy. Patients and Methods: Peripheral blood (PB) was collected from 32 metastatic breast cancer patients and 42 negative controls. The antibodies BM7 and VU1D9 were used for immunomagnetic tumor cell enrichment. A real-time reverse transcription-polymerase chain reaction (RT-PCR) approach for the markers KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 and ERBB2 was used for CTC detection and characterization. Results: The positivity rates for each marker were as follows: 46.9% for KRT19, 25.0% for SCGB2A2, 28.1% for MUC1, 28.1% for EPCAM, 21.9% for BIRC5, and 15.6% for ERBB2. After the creation of individualized cutoffs, the sensitivity and specificity of the combined marker gene panel increased to 56.3% and 100%, respectively. Interestingly, 27.0% of the HER2-negative tumor patients showed ERBB2 mRNA-positive CTCs. Conclusions: The described technique can be used to measure CTCs with great accuracy. The use of a multimarker panel for the characterization of CTCs may provide real-time information and be of great value in therapy monitoring. / Ziel: Entwicklung eines immunomagnetischen Verfahrens zur Isolierung zirkulierender Tumorzellen (CTCs) in Kombination mit einer molekularen Multimarkeranalyse für die hochspezifische Identifizierung maligner Zellen. Patientinnen und Methoden: Peripheres Blut (PB) von 32 Patientinnen mit metastasiertem Mammakarzinom und von 42 gesunden Kontrollen wurde für die immunomagnetische Tumorzellanreicherung mit den Antikörpern BM7 und VU1D9 genutzt. Eine Real-Time Reverse Transkription Polymerase-Kettenreaktion (RT-PCR)-Methodik mit den Markern KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 und ERBB2 wurde für den CTC-Nachweis und die Tumorzellcharakterisierung entwickelt. Ergebnisse: Für die einzelnen Marker wurden die folgenden Positivitätsraten ermittelt: 46,9% für KRT19, 25,0% für SCGB2A2, 28,1% für MUC1, 28,1% für EPCAM, 21,9% für BIRC5 und 15,6% für ERBB2. Nach der Bestimmung individualisierter Cut-off-Werte ergab sich für den kombinierten Multimarkernachweis eine Sensitivität und Spezifität von 56,3% bzw. 100%. Bemerkenswert war der Befund, dass 27,0% der HER2-tumornegativen Patientinnen ERBB2-mRNA-positive CTCs aufwiesen. Schlussfolgerung: Die hier beschriebene Methodik bestimmt CTCs mit hoher Spezifität. Die molekulare Multimarkeranalyse liefert wertvolle Real-Time-Informationen für personalisierte Behandlungsmodalitäten. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.