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Fruit thinning is critical to the success of apple industry because most apple trees are prone to biennial bearing characterized by heavy bloom and over-cropping in the "on" year and low or no cropping in the "off" year. Fruit thinning can improve fruit size, increase return bloom, and reduce alternate bearing habit of apple trees. Chemical thinning has been widely used as a horticultural practice by growers for years in apple and other fruit production. However, its thinning results are often variable and very difficult to predict since parameters as well as their interactions (e.g. concentration, environmental condition, cultivar response) that affect thinning effectiveness have been poorly defined and the regulatory mechanisms at molecular level remain to be illustrated. Therefore, the purpose of this study is to characterize the physiological and molecular responses of the apple trees to the thinning treatments, and then use key genes as molecular markers for screening potential thinning agents. The long-term goal is to understand how the applied chemicals and environmental factors interact and regulate key regulatory genes as well as the thinning effectiveness during thinning process, and establish a predictable model for the improvement of fruit thinning consistency and effectiveness in apple and other fruit trees. Effects of naphthaleneacetic acid (NAA), shading, amimoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP) on young apple fruit abscission, leaf and fruit ethylene production, and the expression of genes related to ethylene biosynthesis, perception and cell wall degradation were examined in "Golden Delicious" apples (Malus x domestica borkh.). NAA at 15 mg·L-1 and shading increased fruit abscission and ethylene production of leaves and fruit when applied at a 12-mm stage of fruit development, whereas AVG, an inhibitor of ethylene biosynthesis, at 250 mg·L-1 reduced NAA-induced fruit abscission and ethylene production of leaves and fruit. 1-MCP at 160 mg·L-1 had no effect on fruit abscission but induced ethylene production by both leaves and fruit. Changes in the gene expression pattern responding to each treatment were analyzed by real-time quantitative PCR. NAA treatment was found to enhance the expression of genes related to ethylene biosynthesis (MdACS5A, MdACS5B and MdACO1) and perception (MdETR1, MdETR1b, MdETR2, MdERS1 and MdERS2). AVG reduced NAA-induced expression of these genes except for MdERS2 in the fruit abscission zone (FAZ). NAA increased the expression of a polygalacturonase gene (MdPG2) in the FAZ but not in the fruit cortex (FC), whereas AVG reduced NAA-enhanced expression of MdPG2 in the FAZ. These results suggest that ethylene biosynthesis, ethylene perception, and MdPG2 gene are involved in young fruit abscission caused by NAA. On the other hand, 1-MCP did not affect the expression of MdACS5A and MdACS5B in the FAZ, although it enhanced the expression of these two genes in the FC from 6 to 24 hours post-treatment. The expression of MdACO1 in both tissues was increased by 1-MCP after 3 or more days post-treatment. 1-MCP had only a small influence on the expression of most ethylene receptor genes, with the exception of MdETR1, which was up-regulated in the FC to a level similar to that observed for NAA treatment. In response to 1-MCP, in the FAZ, the expression of MdCel1 and MdPG2 was up-regulated at the beginning and the end, respectively, of the experiment, but otherwise remained at or below control levels. 1-MCP did not inhibit NAA-induced abscission of young apple fruit, suggesting that abscission does not solely depend on ethylene signal transduction, or that the periods of effectiveness for 1-MCP and ethylene were asynchronous in this study. Gene expression analysis also revealed that both NAA and shading enhanced the expression of gene related to ABA biosynthesis in the FAZ, which suggested the involvement of ABA in young fruit abscission. Global gene expression profile during young fruit abscission was analyzed using an apple oligonucleotide microarray. More than 700 genes were identified with reproducible changes in transcript abundance in the FAZ after NAA treatment. Genes associated with abscission, ethylene, ABA, cell wall degradation, mitochondrial activity, glycolysis, lipid catabolism, secondary metabolism, abiotic stress, and apoptosis were upregulated, while genes involved in regulation of cell cycle, cell wall biosynthesis, photosynthesis, carbon fixation, chromatin assembly, auxin transport/efflux, cytoskeleton function, and flower development were generally downregulated. Comparison of changes in the gene expression patterns during NAA-induced fruit abscission with shading-induced fruit abscission revealed similarities, but also considerable differences. The data suggested that young fruit were shutting down growth and energy production and undergoing a certain kind of programmed cell death (PCD) induced by these different stimuli. Gene expression analysis in abscising young fruit revealed that photosynthesis, carbon utilization, and ABA/ethylene pathways appeared to operate in both NAA- and shading-induced fruit abscission. However, more sugar signaling genes were regulated in shading-induced fruit abscission, compared to NAA-induced fruit abscission. / Ph. D.

A análise da expressão gênica através de dados gerados em experimentos de microarrays de DNA vem possibilitando uma melhor compreensão da dinâmica e dos mecanismos envolvidos nos processos celulares ao nível molecular. O aprimoramento desta análise é crucial para o avanço do conhecimento sobre as bases moleculares das neoplasias e para a identificação de marcadores moleculares para uso em diagnóstico, desenho de novos medicamentos em terapias anti-tumorais. Este trabalho tem como objetivos o desenvolvimento de modelos de análise desses dados, propondo uma nova forma de normalização de dados provenientes de microarrays e dois modelos para a construção de redes regulatórias de expressão gênica, sendo uma baseada na conectividade dinâmica entre diversos genes ao longo do ciclo celular e a outra que resolve o problema da dimensionalidade, em que o número de experimentos de microarrays é menor que o número de genes. Apresenta-se, ainda, um pacote de ferramentas com uma interface gráfica de fácil uso contendo diversas técnicas de análise de dados já conhecidas como também as abordagens propostas neste trabalho. / The analyses of DNA microarrays gene expression data are allowing a better comprehension of the dynamics and mechanisms involved in cellular processes at the molecular level. In the cancer field, the improvement of gene expression interpretation is crucial to better understand the molecular basis of the neoplasias and to identify molecular markers to be used in diagnosis and in the design of new anti-tumoral drugs. The main goals of this work were to develop a new method to normalize DNA microarray data and two models to construct gene expression regulatory networks. One method analyses the dynamic connectivity between genes through the cell cycle and the other solves the dimensionality problem in regulatory networks, meaning that the number of experiments is lower than the number of genes. We also developed a toolbox with a user-friendly interface, displaying several established statistical methods implemented to analyze gene expression data as well as the new approaches presented in this work.

A análise da expressão gênica através de dados gerados em experimentos de microarrays de DNA vem possibilitando uma melhor compreensão da dinâmica e dos mecanismos envolvidos nos processos celulares ao nível molecular. O aprimoramento desta análise é crucial para o avanço do conhecimento sobre as bases moleculares das neoplasias e para a identificação de marcadores moleculares para uso em diagnóstico, desenho de novos medicamentos em terapias anti-tumorais. Este trabalho tem como objetivos o desenvolvimento de modelos de análise desses dados, propondo uma nova forma de normalização de dados provenientes de microarrays e dois modelos para a construção de redes regulatórias de expressão gênica, sendo uma baseada na conectividade dinâmica entre diversos genes ao longo do ciclo celular e a outra que resolve o problema da dimensionalidade, em que o número de experimentos de microarrays é menor que o número de genes. Apresenta-se, ainda, um pacote de ferramentas com uma interface gráfica de fácil uso contendo diversas técnicas de análise de dados já conhecidas como também as abordagens propostas neste trabalho. / The analyses of DNA microarrays gene expression data are allowing a better comprehension of the dynamics and mechanisms involved in cellular processes at the molecular level. In the cancer field, the improvement of gene expression interpretation is crucial to better understand the molecular basis of the neoplasias and to identify molecular markers to be used in diagnosis and in the design of new anti-tumoral drugs. The main goals of this work were to develop a new method to normalize DNA microarray data and two models to construct gene expression regulatory networks. One method analyses the dynamic connectivity between genes through the cell cycle and the other solves the dimensionality problem in regulatory networks, meaning that the number of experiments is lower than the number of genes. We also developed a toolbox with a user-friendly interface, displaying several established statistical methods implemented to analyze gene expression data as well as the new approaches presented in this work.

BACKGROUND:Ginger (Zingiber officinale) and turmeric (Curcuma longa) accumulate important pharmacologically active metabolites at high levels in their rhizomes. Despite their importance, relatively little is known regarding gene expression in the rhizomes of ginger and turmeric.RESULTS:In order to identify rhizome-enriched genes and genes encoding specialized metabolism enzymes and pathway regulators, we evaluated an assembled collection of expressed sequence tags (ESTs) from eight different ginger and turmeric tissues. Comparisons to publicly available sorghum rhizome ESTs revealed a total of 777 gene transcripts expressed in ginger/turmeric and sorghum rhizomes but apparently absent from other tissues. The list of rhizome-specific transcripts was enriched for genes associated with regulation of tissue growth, development, and transcription. In particular, transcripts for ethylene response factors and AUX/IAA proteins appeared to accumulate in patterns mirroring results from previous studies regarding rhizome growth responses to exogenous applications of auxin and ethylene. Thus, these genes may play important roles in defining rhizome growth and development. Additional associations were made for ginger and turmeric rhizome-enriched MADS box transcription factors, their putative rhizome-enriched homologs in sorghum, and rhizomatous QTLs in rice. Additionally, analysis of both primary and specialized metabolism genes indicates that ginger and turmeric rhizomes are primarily devoted to the utilization of leaf supplied sucrose for the production and/or storage of specialized metabolites associated with the phenylpropanoid pathway and putative type III polyketide synthase gene products. This finding reinforces earlier hypotheses predicting roles of this enzyme class in the production of curcuminoids and gingerols.CONCLUSION:A significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric. Specific transcription factors and other regulatory genes were found that were common to the two species and that are excellent candidates for involvement in rhizome growth, differentiation and development. Large classes of enzymes involved in specialized metabolism were also found to have apparent tissue-specific expression, suggesting that gene expression itself may play an important role in regulating metabolite production in these plants.

The airway epithelium demonstates the ability to quickly repair following physical injury. The morphologic features of this dynamic repair process have been well characterised at the anatomic and cellular level using a number of animal model systems and these studies have provided a solid foundation upon which our understanding of normal repair is build. With the advent of molecular and bioinformatic tools and resources the opportunity exists to extend the value of these models in defining the molecular pathways and interactions that underlay the normal repair process. This thesis represents a realisation of this opportunity. A large animal model was developed in which selected areas of airway epithelium were subjected to bronchial brush biopsy as part of routine bronchoscopic examination prcedures in anaethetised sheep. The process resulted in a physical perturbation of the normal pseudostratified structure of the sheep airwway epithelium at specific locations. By careful experimental design it was possible, within the same animals. to identify and sample from sites undergoing repair at different intervals subsequent in injury. To supplement the histological evaluation of the repair process and align findings with extablished small animal models of airway epithelial repair proliferative cell labelling strategies were implemented in order to study the location and extent of cellular proliferation occurring duringthe repair process. Molecular approaches towards defining the transcriptional response to physical injury comprised application of microarray technology using a commercially sources array platform. Such approach demanted preliminary effort directed towards optimising RNA integrity and yield from airway samples. Following preliminary studies directed towards optimising the model conditions patterns of airway epithelial repair following bronchial brush biopsy were studies in eight sheep at degined time points (6 hours, 1,3, & 7 days) post-injury. Bronchial brush biopsy resulted in the acute removal of the epithelial cell layer and components of the underlying structures. repair processes were rapidly implemented through initial epithelial dedifferentiation, proliferatino and migration at the wound margins and subsequent time-depentend changes in the proportion of subepithelial structures, including smooth muscle and blood vessels, as the epithelial surface moved towards repair. Transcriptional analysis revewaled that over 13,000 probes showed evidence of differential expession at some point during the repair process (p<0.05), whilst of these, 1491 probes had in excess of a two-fold change in expression. array results were validated against conventional semi-quantitative RT-PCR for selected genes. Differentially expressed genes with previously characterised roles in epithelial migration, prolifereation and differentiation were identified during the repair process. The relative emphasis of gene products with particular functional roles varied during the course of repair. Indeed gene ontology (GO) terms identified included those associated with the inflammatory response, cellular migration, extracellular matrix activities, differentiation, proliferation, cellular development, cell cycle activities, cellular adhesion, apoptosis and mitosis. In addition the Kyoto Encyclopedia of Genes and Gneomes (KEGG) databases were queried and such process indicated the involvement of cell communication, 053 and complement and coagulation cascade pathways throughout the repair process, initial (6h) Toll-like receptor and cytokine-cytoine receptor interaction pathways, and the progressive involement of cell cycle, focal adhesion and extracellulaar matrix (ECM)-receptor, and cytokine interaction pathways as the epithelium repaired. The model of airway epithelial injury developed in this thesis generated features broadly consistent with those previosly described in relation to various small animal model systems. Importantly, and in addition, this thesis defines the molecular features associated with repair in this model system and provides a useful resource with which to assess the comparative fetures of the airway transcriptional response to physical injury, It is through such comparison, using analogous methodology, that the fundamental pathways and interactions that underlay normal repair and regeneration can be identified and therafter extended towards inderstanding the basis for variation associated with natural and experimental disease.

Sepsis-related multiple organ dysfunction syndrome is a leading cause of death in intensive care units. There is overwhelming evidence that oxidative stress plays a significant role in the pathogenesis of sepsis-associated multiple organ failure; however, reactive oxygen species (ROS)-associated biomarkers and/or diagnostics that define mortality or predict survival in sepsis are lacking. Lung or peripheral blood gene expression analysis has gained increasing recognition as a potential prognostic and/or diagnostic tool. The objective of this study was to identify ROS-associated biomarkers predictive of survival in patients with sepsis. In-silico analyses of expression profiles allowed the identification of a 21-gene ROS-associated molecular signature that predicts survival in sepsis patients. Importantly, this signature performed well in a validation cohort consisting of sepsis patients aggregated from distinct patient populations recruited from different sites. Our signature outperforms randomly generated signatures of the same signature gene size. Our findings further validate the critical role of ROSs in the pathogenesis of sepsis and provide a novel gene signature that predicts survival in sepsis patients. These results also highlight the utility of peripheral blood molecular signatures as biomarkers for predicting mortality risk in patients with sepsis, which could facilitate the development of personalized therapies.

The analysis techniques applied to DNA microarray images are under active development. As new techniques become available, it will be useful to apply them to existing microarray images to obtain more accurate results. The compression of these images can be a useful tool to alleviate the costs associated to their storage and transmission. The recently proposed Relative Quantizer (RQ) coder provides the most competitive lossy compression ratios while introducing only acceptable changes in the images. However, images compressed with the RQ coder can only be reconstructed with a limited quality, determined before compression. In this work, a progressive lossy-to-lossless scheme is presented to solve this problem. First, the regular structure of the RQ intervals is exploited to define a lossy-to-lossless coding algorithm called the Progressive RQ (PRQ) coder. Second, an enhanced version that prioritizes a region of interest, called the PRQ-region of interest (ROI) coder, is described. Experiments indicate that the PRQ coder offers progressivity with lossless and lossy coding performance almost identical to the best techniques in the literature, none of which is progressive. In turn, the PRQ-ROI exhibits very similar lossless coding results with better rate-distortion performance than both the RQ and PRQ coders.

Inhibition of disulfide bond formation in Escherichia coli implicates an intricate collaboration of proteins which comprise the glutathione and thioredoxin reducing pathways. Bioengineers have successfully engineered E. coli possessing mutated reducing pathways that promote, rather than inhibit, disulfide bond formation in the cytoplasm. The transcriptome of six such mutant E. coli strains have been characterized using Microarray technology. We find that all mutant strains, exhibit a unique response to oxidative stress, not observed in wild type. Statistical analyses revealed the expression of more than 200 genes that are affected by mutations within the reducing pathways. Significantly up-regulated biological processes include cysteine biosynthesis, histidine biosynthesis, NADH Dehydrogenase I biosynthesis, sugar catabolic processes, and activation of stress responses . The second part of this work describes the construction of an E. coli strain that promotes the complete conversion of glutathione into its seemingly dormant derivative, glutathionylspermidine. This engineered strain can be used in assays designed to evaluate the effectiveness of glutathionylspermidine as a substitute for glutathione and, hopefully, allude to its true metabolic function. / text