Dissertations / Theses on the topic 'Phenotype Microarry'

Breast cancer is the commonest malignancy to affect women and approximately 1 in 9 women (12 %) will develop the disease in developed countries (Burstein and Winer 2000). A major problem for treatment is the development of a drug resistant phenotype, whereby the tumour fails to respond to chemotherapy. This phenotype arises from altered gene expression in the resistant cells, which can occur by changes in transcription and/or genomic alteration. Other phenotypic properties of breast carcinoma cells may also arise in this way, and gene expression profiles can be linked to different breast tumour phenotypes, such as oestrogen receptor (ER) status, clinical tumour stage and tumour size (Martin et al., 2000). In this thesis cDNA microarrays were utilised to study both genomic amplification and RNA expression changes occurring in human breast carcinoma. These changes were related to phenotypic characteristics including a doxorubicin (Dox) resistant phenotype, hormone receptor status, tumour grade and type. Several gene clusters involving the development of resistance and the eventual Dox resistant phenotype in breast cancer cell lines were elucidated, associated with both these was the multi drug resistance 1 gene (ABCB1). Potential therapeutic targets in these cells e.g. the oxytocin receptor gene (OXTR) were also indicated. Regions of genomic amplification and specific genes were elucidated some of these have previously been described while others are novel. Some of the genomic changes were associated with tumour phenotype, for example gene amplification at chromosome 2p25 and 22qll were specific to lymph node positive tumours and 2 genes were consistently found amplified in these regions, LPIN1 and DGSI respectively. The findings of this study have contributed to the general understanding of genetic events occurring in breast cancer and associations between these changes and phenotype were suggested.

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.

Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog((R)) phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant-and seed fungus interactions, such as D-trehalose, myoinositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusariurn species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.

Thesis (Ph. D.)--Ohio State University, 2005.
Document formatted into pages; contains xxi, 216 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 9.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.
Includes bibliographical references (leaves 197-213).
Corynebacterium glutamicum partial genome DNA microarrays were constructed that were capable of assaying the transcriptional profile of the genes of pathways involved in central carbon metabolism and lysine biosynthesis. It was found that to ensure arrays of high quality, protocols applying the arrays should include DNase treatment of RNA samples. additional RNA filtration purification steps, and the use of gene specific primers in the formation of labeled cDNA through reverse transcription. After implementing these procedures, the accuracy and reproducibility of the array data were validated. The microarrays were used to explore the effects of the over-expression of the key anaplerotic enzyme pyruvate carboxylase and the use of different medium carbon source compositions, both of which have been shown to influence the yields of biomass on carbon and of lysine on biomass. Three different strains of C. glutamicum that were grown on six different minimal medium formulations that varied in their balance of glucose and lactate were assayed by isolating total mRNA samples from cultures in three different phases of growth and lysine production. Genes associated with glycolysis and the pentose phosphate pathway showed decreased transcript concentrations as the available carbon source was shifted from glucose to lactate, while those associated with the TCA cycle and the glyoxylate bypass demonstrated increased transcription. As the cultures stopped generating biomass and began generating lysine, mRNA of genes associated with lysine synthesis and export was measured at elevated concentrations.
(cont.) Reduced gene expression trends seen for aspartokinase and aspartate semialdehyde dehydrogenase suggest that the enzymes are bottlenecks to lysine production, particularly when pyruvate carboxylase is over-expressed and lactate is the available carbon source. This over-expressing strain also had higher transcription levels of the genes of biotin synthesis. and lower transcription levels of the acyl-coA carboxylases dtsRl, dtsR2, accC, and accD. Other results implied that malic enzyme is co-expressed with pyruvate carboxylase to better allow cultures grown on lactate to produce NADPH in the absence of significant pentose phosphate pathway flux. Also, the transcriptional and flux profiles of a pair of C. glutamicum strains grown on two different medium compositions of isotopically labeled glucose and lactate were determined simultaneously from the same set of actively growing and lysine-producing cultures. Flux maps for each of the four combinations of strain and medium were constructed using calculations derived from metabolite balances and GC-MS measurements of the isotopic distributions within biomass hydrolysates of the pseudo-steady-state cultures. Comparisons of the two sets of data showed that 19 of 28 pairs of flux and transcription measurements had trends with good agreement with one another. Different pathways of the metabolic network were found to be controlled via transcription in varying degrees. On average, the Embden- Meyerhof-Parnas pathway was shown to be less likely to be regulated though transcription than the pathways of the tricarboxylic acid cycle and central carbon anaplerosis.
(cont.) In the split pathway available to the cells for producing lysine, the succinylation branch showed an increase in flux for only the case of a pyruvate carboxylase over-expressing strain that was grown on lactate, while the alternate dehydrogenation branch showed a complementary decrease in flux. These flux changes were matched by changes in transcription that only occurred for the same culture and growth medium. Through these findings we have demonstrated the application of C. glutamicum DNA microarrays to the determination of how the cells regulate their responses at the transcriptional level to changes in both gene over-expression and medium composition.
by Christopher Roberge.
Ph.D.

The past decade has witnessed significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in a large number of genes have now been implicated in the pathogenesis of MPN but their relative importance, the mechanisms by which they cause different cells to predominate and differentiate into the separate MPN syndromes and their implications for prognosis remain unknown. I hypothesized that other genes and epigenetic mechanisms may contribute to the pathogenesis of the different disease subtypes at a cell-type specific level. This study focused on four areas: (1) Clinical phenotype - The demographic and clinical landscape of MPN in Malaysia showed that MPN is predominantly found in older Malay men. Compared to other studies, there is a higher incidence of JAK2 V617F positivity, with a more severe clinical phenotype and complications. (2) MPN panel - JAK2 V617F is the commonest somatic driver mutation detected in MPN patients using this 26-gene MPN targeted sequencing panel. The highest allelic frequencies were found in polymorphonuclear (PMN) and mononuclear cells (MNC). Several novel variants were found. Primary myelofibrosis patients who harbor the JAK2 V617F in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 putatively pathogenic variants were found to have more severe clinical phenotypes and poor prognosis. (3) Gene Expression and (4) DNA methylation - Using microarray and next generation sequencing techniques, cell type-specific differentially expressed genes and differentially methylated CpG sites were found in PMN and MNC in the different disease subtypes. The lack of differential expression and methylation in T cells validated the approach and indicated they are not part of the neoplastic clone. The differentially expressed genes and differentially methylated CpG sites with associated genes are candidate loci to explain the pathogenesis of MPN and its different forms. These loci also represent targets for further investigation and disease-specific therapeutic targets.

Asthma is a respiratory syndrome associated with airflow limitation, bronchial hyperresponsiveness and inflammation of the airways in the lungs. Despite the ongoing research efforts, the outstanding heterogeneity displayed by the multiple forms in which this condition presents often hampers the attempts to determine and classify the phenotypic and endotypic biological structures at play, even when considering a limited assembly of asthmatic subjects. To increase our understanding of the molecular mechanisms and functional pathways that govern asthma from a systems medicine perspective, a computational workflow focused on the identification of personalized transcriptomic modules from the U-BIOPRED study cohorts, by the use of the novel MODifieR integrated R package, was designed and applied. A feature selection of candidate asthma biomarkers was implemented, accompanied by the detection of differentially expressed genes across sample categories, the production of patient-specific gene modules and the subsequent construction of a set of core disease modules of asthma, which were validated with genomic data and analyzed for pathway and disease enrichment. The results indicate that the approach utilized is able to reveal the presence of components and signaling routes known to be crucially involved in asthma pathogenesis, while simultaneously uncovering candidate genes closely linked to the latter. The present project establishes a valuable pipeline for the module-driven study of asthma and other related conditions, which can provide new potential targets for therapeutic intervention and contribute to the development of individualized treatment strategies.

L'endocardite infectieuse (EI) est une complication rare mais gravissime de la bactériémie à Staphylococcus aureus. Bien que certains facteurs de risque liés à l'hôte aient été décrits, l'implication de facteurs bactériens dans la survenue de l'EI est encore inconnue. Ces travaux de thèse ont visé à chercher tout élément bactérien associé à l'EI. Les facteurs phénotypiques décrits ou supposés comme potentiellement impliqués dans l'EI ont été testés. En parallèle, les profils génotypiques des souches obtenus par puces ADN ont été analysés par différents outils statistiques. L'analyse statistique univariée n'a montré aucune différence significative entre souches d'EI et souches de bactériémie, suggérant un processus complexe et multifactoriel. En effet, l'analyse discriminante en composante principale appliquée sur les données de puces ADN a permis de mettre en évidence une distinction entre les deux groupes de souches, confirmée sur une collection indépendante de souches. De plus, une fonction linéaire simplifiée, basée sur seulement 8 marqueurs génétiques, a permis d'obtenir des performances similaires, sur la collection de souches initiale ainsi que la collection indépendante de validation. En dernier lieu, les souches d'EI et de bactériémie ont été comparées à partir de séquences du génome complet (n = 40 (20 EI, 20 bactériémies)). L'analyse statistique par analyse discriminante en composante principale réalisée sur ces données génomiques confirme une distinction possible entre les deux groupes de souches. Au total, ces travaux de thèse apportent la preuve de concept que les facteurs bactériens sont impliqués dans la survenue de l'EI au cours de bactériémie à S. aureus
Infective endocarditis (IE) is a severe condition complicating 10-25% of Staphylococcus aureus bacteremia. Although host-related IE risk factors have been identified, the involvement of bacterial features in IE complication is still unclear. This PhD work aimed to characterize strictly defined IE and bacteremia isolates and searched for discriminant features. Phenotypic traits previously reported or hypothesized to be involved in staphylococcal IE pathogenesis were tested. In parallel, the genotypic profiles of all isolates, obtained by microarray, were analyzed. No significant difference was observed between IE and bacteremia strains, regarding either phenotypic or genotypic univariate analyses, suggesting a multifactorial process. However, the discriminant analysis of principal components (DAPC), applied on microarray data, segregated IE and bacteremia isolates. The performance of this model was confirmed with an independent collection of IE and bacteremia isolates. Finally, a simple linear discriminant function based on a subset of 8 genetic markers retained valuable performance both in study collection and in the independent validation collection. At last, IE and bacteremia isolates were compared based on whole genome sequence data from a subset of 40 isolates. When applied to this dataset, DAPC confirmed a possible segregation between the two groups of isolates. All in all, this PhD work provides the proof of concept that bacterial characteristics may contribute to the occurrence of IE in patients with S. aureus bacteremia

La thèse s’inscrit dans un projet de recherche global visant à caractériser les tumeurs thyroïdiennes sur le plan moléculaire, afin de mieux comprendre leur physiopathologie et afin d’identifier des biomarqueurs (signatures moléculaires) qui pourront être utilisés pour le diagnostic, le pronostic et leur traitement. Parmi celles-ci, nous distinguons les adénomes autonomes (AA) et folliculaires (FTA) tumeurs bénignes encapsulées, et les carcinomes, tumeurs malignes. Ceux-ci sont eux-mêmes subdivisés en carcinomes différenciés, folliculaires (FTC) ou papillaires (PTC), et peuvent évoluer en carcinomes anaplasiques (ATC), totalement dédifférenciés. Un autre type de tumeurs bénignes différenciées, très rares, existe: l’hyperthyroïdie non auto-immune familiale (FNAH). Ces tumeurs sont causées pour la plupart par des mutations qui activent de manière constitutive des cascades de signalisation, essentiellement la cascade de l’AMPc et la cascade des MAPK. Le but de notre thèse était de valider un système expérimental in vitro des PTC, d’étudier les profils d’expression génique des FNAH et de les comparer avec ceux des AA, et de définir les profils ARNm et miRNA des ATC pour les comparer à ceux des PTC pour identifier de nouvelles cibles thérapeutiques potentielles. Pour réaliser ces objectifs, nous avons utilisé la technologie des microarrays qui permet d’analyser simultanément l’expression de milliers de gènes dans différentes conditions. Nous avons donc utilisé une approche multidisciplinaire alliant une partie expérimentale et une partie bioinformatique.

La première partie du travail a consisté à réaliser un modèle d’étude in vitro pour caractériser les PTC au niveau moléculaire. A cet effet, des cultures primaires de thyrocytes ont été traitées avec de l’EGF et du sérum pendant différents temps (1,5h, 3h, 16h, 24h et 48h) ce qui stimule la cascade des MAPK, activée constitutivement dans les PTC. Nous avons hybridé sur des lames microarrays maison les différents échantillons et nous avons montré que les cultures primaires stimulées pendant des temps longs (24h et 48h) ont des profils d’expression génique qui ressemblent à ceux des PTC et constituent donc un bon modèle d’étude de cette tumeur.

La seconde partie a pour objectif de définir les phénotypes moléculaires et fonctionnels des FNAH et de les comparer aux AA. Ces deux pathologies résultent d’une mutation dans le récepteur de la TSH (TSHR) activant de manière constitutive la cascade de l’AMPc. Dans le cas des FNAH, la mutation est héréditaire et toute la glande est affectée contrairement aux AA où la mutation survient plus tard, généralement à l’âge adulte, et où seule une partie de la glande est affectée. Nous avons comparé le profil d’expression génique des FNAH avec celui des AA, par hybridation sur des lames microarrays HEEBO. L’intégration de ces différentes données montre que les AA et les FNAH sont deux sous-types différents de la même maladie: l’hyperthyroïdie génétique. Les caractéristiques de chacun de ces sous-types dépendent de l’intensité de la mutation, du nombre de cellules initialement affectées et du stade de développement au moment duquel la mutation survient.

Dans la dernière partie de ce travail, nous avons caractérisé les ATC au niveau du profil d’expression des ARNm et des miRNA par hybridation respectivement sur lames Affymetrix ou sur lames miRNA maison et au niveau de leur état mutationnel du gène p53. Le profil d’expression génique des ATC a été comparé avec celui des PTC afin de mettre en évidence des gènes différentiellement exprimés entre les 2 types de cancers, que nous avons ensuite tenté d’invalider par siRNA, dans un modèle in vitro de lignée cellulaire thyroïdienne dérivée d’un ATC (8505C). Les résultats obtenus jusqu’ici ne sont malheureusement pas prometteurs. Le profil d’expression des miRNA nous a permis d’identifier une signature de 34 miRNA caractéristique des ATC.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Nucleotide based second messengers are known to regulate wide variety of processes in all domains of life. Two such bacterial second messengers are (p)ppGpp (guanosine tetra- or pentaphosphate) and c-di-GMP (cyclic dimeric guanosine monophosphate). The alarmone (p)ppGpp is synthesized by bacteria to face any kind of stress; while the signalling nucleotide c-di-GMP is synthesized principally to switch from motile (planktonic) to sessile (biofilm) life style. Apart from mediating the said functions, these nucleotides also regulate transcription, translation, replication, virulence and pathogenicity of the several bacterial species. In this work, we have tried to uncover novel functions or phenotypes that are governed by the second messengers (p)ppGpp and c-di-GMP in Mycobacterium smegmatis. In M. smegmatis, (p)ppGpp and c-di-GMP are synthesized and degraded by the bifunctional proteins RelMsm and DcpA, respectively. The architecture of both the proteins is similar; the synthesis and hydrolysis domains for the second messengers occur in tandem. The knockout mutants of relMsm and dcpA genes, ∆relMsm and ∆dcpA, have been used in this study to uncover the novel functions of these second messengers in mycobacterial physiology. Chapter 1 provides is an overview of the current literature pertaining to (p)ppGpp and c-di-GMP. An historical perspective with regard to the discovery of the (p)ppGpp and c-di-GMP is given. The metabolism of these second messengers has been discussed. This has been followed by the description of various functions governed by the second messengers. Finally, the scope of the current work has been outlined. Chapter 2 investigates the effect of disrupting (p)ppGpp and c-di-GMP signalling on the antibiotic sensitivity in M. smegmatis. Using Phenotype Microarray (PM) technology, the growth of ∆relMsm and ∆dcpA knock out strains was compared to those of the wild-type and respective complemented strains in 240 different antimicrobials. It was found that the knockout mutants displayed enhanced survival in the presence of multiple antibiotics. The PM data was corroborated by the independent determination of minimum inhibitory concentrations of seven different antibiotics. Finally, the plausible reasons for the multidrug resistance of ∆relMsm and ∆dcpA strains have been discussed. Chapter 3 explores how the impairment of (p)ppGpp and c-di-GMP alters the cell wall of M. smegmatis. Thin layer chromatography analysis of cell wall fractions such as glycopeptidolipids (GPLs), mycolic acids, polar and apolar lipids was carried out. It was found that the amount of GPLs and polar lipids were reduced in the ∆relMsm and ∆dcpA knockout strains. Chapter 4 explores the effect of (p)ppGpp and c-di-GMP on the growth, cell morphology and cell division in M. smegmatis. It was found that the ∆relMsm and ∆dcpA knockout strains have slow growth compared to those of the wild type and respective complemented strain. The overproduction of (p)ppGpp and c-di-GMP, achieved through overexpression of Rel and DcpA proteins, encased the overexpression strains relOE and dcpAOE in a biofilm like matrix. The higher levels of (p)ppGpp and c-di-GMP caused M. smegmatis assume coccoid morphology. Microscopy analyses revealed that the ∆relMsm and ∆dcpA strains are elongated, multinucleate and multiseptate. Chapter 5 explores effects of (p)ppGpp and c-di-GMP on the global gene expression profile in M. smegmatis. Many genes were shown to be differentially expressed in the ∆relMsm and ∆dcpA knockout strains. Genes regulating cell division, cell wall biosynthesis, superoxide metabolism or reactive oxygen species metabolism and genes encoding transporters were differentially expressed in the ∆relMsm and ∆dcpA knockout mutants. The microarray data were corroborated by quantitative real-time PCR. Gene expression data explained the multidrug resistance, the reduction in the level of GPLs and polar lipids, slow growth, changes in cell morphology and defective cell division exhibited by the ∆relMsm and ∆dcpA knockout mutants. Chapter 6 summarizes the entire work embodied in the thesis. Appendix 1 lists the 240 antimicrobials compounds and their mode of action for which antibiotic sensitivity of the ∆relMsm and ∆dcpA knockout mutants was tested. Appendix 2 lists the growth differences among the knockout, wild type and complemented strains in the form of area under curve values. Appendix 3 lists the genes that were differentially expressed in the ∆relMsm and ∆dcpA knockout strains. Appendix 4 is a comprehensive review on the kinetic and thermodynamic parameters governing the sigma factor competition in Escherichia coli and how (p)ppGpp and anti-sigma factors regulate this competition among sigma factors for the limited pool of core RNA polymerase in E. coli.

The past few decades have witnessed a revolution in recombinant DNA and nucleic acid sequencing technologies. Recently however, technologies capable of massively high-throughout, genome-wide data collection, combined with computational and statistical tools for data mining, integration and modeling have enabled the construction of predictive networks that capture cellular regulatory states, paving the way for ‘Systems biology’. Consequently, protein interactions can be captured in the context of a cellular interaction network and emergent ‘system’ properties arrived at, that may not have been possible by conventional biology. The ability to generate data from multiple, non-redundant experimental sources is one of the important facets to systems biology. Towards this end, we have established a novel platform called ‘spotted cell microarrays’ for conducting image-based genetic screens. We have subsequently used spotted cell microarrays for studying multidimensional phenotypes in yeast under different regulatory states. In particular, we studied the response to mating pheromone using a cell microarray comprised of the yeast non-essential deletion library and analyzed morphology changes to identify novel genes that were involved in mating. An important aspect of the mating response pathway is large-scale spatiotemporal changes to the proteome, an aspect of proteomics, still largely obscure. In our next study, we used an imaging screen and a computational approach to predict and validate the complement of proteins that polarize and change localization towards the mating projection tip. By adopting such hybrid approaches, we have been able to, not only study proteins involved in specific pathways, but also their behavior in a systemic context, leading to a broader comprehension of cell function. Lastly, we have performed a novel metabolic starvation-based screen using the GFP-tagged collection to study proteome dynamics in response to nutrient limitation and are currently in the process of rationalizing our observations through follow-up experiments. We believe this study to have implications in evolutionarily conserved cellular mechanisms such as protein turnover, quiescence and aging. Our technique has therefore been applied towards addressing several interesting aspects of yeast cellular physiology and behavior and is now being extended to mammalian cells.
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碩士
國立臺灣大學
工業工程學研究所
92
Microarrays are widely used to monitor gene expressions to yield information for genomes. Though there are many methods and mechanisms proposed to extract information from microarray data, the preprocess of raw expression data determine the accuracy and reliability of the extracted information. The first objective of this research is to implement a systematic procedure to preprocess the raw intensity reading. The proposed data preprocess procedure has 3 steps: rectification of intensity reading, signal normalization and bad spots screening. The rectification of intensity uses coefficient of variation (CV) to assess the consistencies of mean intensity and median intensity from raw intensity readings to decide which one to employ and then test the correlations between foreground intensity and background intensity to correct background intensity effects. Signal normalization transforms the rectified data to remove the chip-to-chip brightness variation and contrast variation by logarithm transformation, median subtraction and deviation division. After signal normalization, the hypothesis T-test is used to screen out bad expressions in replicated spots. More recently, microarrays have been conducted not only to relate genes with one phenotype, but also inquire relations between gene expression levels and multiple phenotypes. The second objective of this research is to apply Factor Analysis (FA) to extraction of the underlying co-regulating and independent factors of the multiple phenotypes. And then the treated factors can be taken as an individual phenotype for testing differentially expressed genes. Both of the objectives are to prepare experimental readings for accurate, effective biological information mining procedure. Finally, a real case of microarray experiment investigating gene expressions in 24 human blood samples with 19 phenotypes is provided to demonstrate and test the proposed preprocessing procedures.

Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.

Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification; however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.

Associations between leguminous plants and symbiotic nitrogen-fixing bacteria (rhizobia) are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Rhizobia improve plant yield furnishing fixed nitrogen; therefore, they are highly used as inoculants, especially in sustainable agriculture. Though this association is species-specific, legume roots are exposed to heterogeneous rhizobial populations where different compatible strains are present and, could be infected by more than one strain. It is known that within the same rhizobial species different strains may have different competition capabilities, but detailed analyses able to predict the rhizobial competitive phenotype based exclusively on their genome are still lacking. In this thesis, we performed a bacterial genome-wide association analysis (GWAS) to define which genetic traits are responsible for an improved competitive phenotype in the model species Sinorhizobium meliloti. A panel of thirteen S. meliloti strains, whose genome is completely sequenced, has been selected and tested against three S. meliloti reference competitor strains (Rm1021, AK83 and BL225C) in a Medicago sativa nodule occupancy test. The measure of competition phenotypes previously obtained in the competition tests, in combination with strains genomic sequences of strains tested, were used to build-up a k-mer-based statistical models for each set of competition experiments. The obtained models were then applied to evaluate the accuracy in predicting the competition abilities of strains in the three competition patterns (vsRm1021, vsAK83 and vsBL225C). The competitive abilities of S. meliloti strains against two partners, BL225C and Rm1021, were well predict by the predictive models, as shown by the coefficient of determination R2 (equal to 0.96 and 0.84, respectively). Four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) versus BL225C were used to identify k-mers associated with the competition phenotype. The most significantly associated k-mers (p <0.05) were mapped on the genomic sequences of the S. meliloti strains used. Most of the k-mers were located on the symbiosis-related megaplasmid pSymA and on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (i.e. glycerol, fatty acids) suggesting that competition abilities reside in multiple genetic determinants comprising several cellular components. The identification of the best rhizobial inoculants in two new effective breeding lines of pea (Pisum sativum) used in Lithuania (DS 3637-2 and DS 3795-3) was also investigated. Six rhizobial strains, isolated from pea plants, which could be used as potential inoculants, were phylogenetically identified and extensive phenotypically characterized by Phenotype Microarray. All the strains belonged to the Rhizobium leguminosarum group, and were subdivided into three groups related to Rhizobium anhuiense, Rhizobium leguminosarum bv. viciae and R. sophorae/R. laguerreae. Differences observed with Phenotype microarray were linked to different phylogeny of the strains. In terms of symbiotic efficiency, six strains showed different symbiotic performances depending on the breeding line used. In particular, Rhizobium anhuiense strain Z1 (the reference strain) and Rhizobium leguminosarum bv. viciae 14ZE were the best symbiotic inoculants with breeding lines DS 3637-2 and DS 3795-3, respectively.

Nas últimas décadas, as bactérias multiresistentes tornaram-se numa das principais ameaças à saúde global, desenvolvendo mecanismos e estratégias para ultrapassar os mais recentes avanços da ciência. A família das Enterobacteriaceae conta com vários exemplos de espécies multiresistentes, entre as quais a espécie Klebsiella pneumoniae, um patogénio nosocomial oportunístico, causador de infecções associadas aos cuidados de saúde, com grande capacidade de adaptação a diferentes ambientes e resistência a um grande leque de terapias. Estes agentes patogénicos adquirem resistência das mais variadas formas, quer acidentalmente através de mutações espontâneas como também podem alterar os níveis de produção e as funcionalidades de certos genes. Estas alterações podem influenciar exclusivamente o alvo de determinadas drogas como podem afectar todo um locus e consequentemente um leque de genes e/ou proteínas dependentes deste. A aquisição de resistência através da alteração de genes e/ou proteínas reguladoras é de especial interesse, pois raramente estes factores de transcrição servem unicamente para controlar os níveis de resistência, intervindo e controlando também o metabolismo e fisiologia bacteriana, podendo assim ser apresentados como alvos alternativos para terapias inovadoras. A proteína RamA, presente na espécie Klebsiella pneumoniae e noutros membros da família Enterobacteriaceae é um factor de transcrição com um papel na resistência a antibióticos e no controlo da virulência. A sobreexpressão desta proteína confere um fenótipo multiresistente aos microrganismos. Este projecto tem como objectivo compreender a influência de RamA no desenvolvimento de resistência a antibióticos e ao nível do metabolismo, através do estudo da sua regulação e expressão. Outro objectivo é compreender como actuam estes factores de transcrição como reguladores globais, afectando outras vias ou mecanismos não relacionados com a resistência a antibióticos e/ou virulência. O estudo da função do locus ram no panorama global da emergência de resistência aos antibióticos é de extrema importância visto que, ao contrário da maioria dos mecanismos de resistência, este processo não é quimicamente específico, mas sim uma resposta aos mais variados compostos. Estudar os compostos indutores deste locus e o seu impacto na expressão de ramA e ramR são muito importantes na compreensão do seu papel na adaptação das bactérias a novos ambientes e desafios. Numa primeira fase, foram identificados compostos que têm impacto no crescimento de estirpes mutantes isogénicas que sofreram deleções nos genes ramA e ramR. Para a identificação dos compostos procedeu-se à realização de “phenotypic microarrays” (PM), onde verificámos o impacto da delecção dos genes ramA e ramR no crescimento, sendo observado diferentes padrões de crescimento. Numa tentativa de reproduzir os resultados alcançados realizámos curvas de crescimento em meio apropriado, suplementado com compostos onde o crescimento de um mutante foi superior ao outro. O insucesso na reprodução dos resultados levou-nos a utilizar placas produzidas pelo mesmo fabricante dos PM e com um modus operandi semelhante mas específicas para bactérias gram-negativas, as placas GN. Alcançada a reprodução dos resultados com sucesso, foi necessário verificar qual dos genes, ramA ou ramR, era responsável pelos padrões de crescimento distintos. Estirpes recombinantes e respectivos controlos foram utilizadas para esse efeito. Os resultados acabaram por mostrar que o gene ramR parecia ter mais influência no metabolismo e crescimento do que o gene ramA, levando à formulação da hipótese de que o gene ramR poderia ter um papel de regulador global e não só de repressor local de ramA. Os resultados também indicaram que ramR parecia influenciar o metabolismo de açúcares e fontes de carbono, compostos estes que foram relatados como fazendo parte das cadeias biosintéticas de estruturas membranares na bactéria Escherichia coli. Estes novos dados e as observações de alterações fenotípicas nas estirpes recombinantes levaram-nos a considerar a hipótese de RamR poder controlar a expressão de estruturas membranares, como a membrana de lipopossacarídeos (LPS). No entanto, o estudo dos perfis de LPS das diferentes estirpes envolvidas neste trabalho não mostrou nenhuma diferença significativa entre elas, deixando aberta a possibilidade de realmente a camada de LPS não ser afectada de algum modo e serem outras estruturas membranares as responsáveis pelos fenótipos observados. Numa segunda fase testámos os efeitos de dois compostos, não presentes no PM, que se sabiam ter efeito sobre o locus ram no género Salmonella. Estes compostos, clorpromazina (CPZ) e indol, foram adicionados ao meio apropriado para a realização das curvas de crescimento. As curvas de crescimento na presença de CPZ não confirmaram ou contrariaram a literatura existente, obtendo resultados muito especulativos. A adição de indol ao meio nas curvas de crescimento não alterou de forma alguma o desenvolvimento das estirpes mutantes estudadas. Por meio de outros estudos sabe-se que estes dois compostos actuam de forma semelhante, a nível molecular, no locus ram de Salmonella e Klebsiella, mas isso não se reflecte ao nível do crescimento. Este estudo demonstra principalmente que o locus ram não é constítuido por apenas um regulador global, sugerindo a importância de ramR no crescimento e metabolismo bacteriano. Confirmando-se o envolvimento de RamR com diversas estruturas, responsáveis por características como a virulência ou patogenicidade, ganha-se um potencial alvo para novas terapias. O estudo do locus ram revela-se, assim, de extrema importância para compreender totalmente o seu envolvimento na aquisição da multiresistência por parte da bactéria K. pneumoniae e assim poder encontrar maneiras inovadoras de controlar um problema crescente para a saúde humana.

Η θερμοκρασία ανάπτυξης αποτελεί παράγοντα μεγάλης σημασίας στην οντογένεση των ιχθύων, αφού ως ποικιλόθερμοι οργανισμοί είναι συνεχώς εκτεθειμένοι στις μεταβολές του περιβάλλοντός τους. Έχει παρατηρηθεί πως η θερμοκρασία ανάπτυξης δύναται να προκαλέσει μετατόπιση του χρονοδιαγράμματος των οντογενετικών γεγονότων και πλαστικότητα σε μορφολογικούς και φυσιολογικούς χαρακτήρες (πχ στο μυοσκελετικό και το καρδιαγγειακό σύστημα). Ωστόσο, μέχρι σήμερα δεν έχει μελετηθεί η επίδραση της θερμοκρασίας ανάπτυξης στο πρότυπο της γονιδιακής έκφρασης του zebrafish και σκοπός της παρούσας εργασίας είναι η μελέτη του ολικού μεταγραφικού προτύπου νυμφών zebrafish. Για το λόγο αυτό σχεδιάστηκαν δύο πειράματα, όπου τρεις θερμοκρασιακές συνθήκες ανάπτυξης (22, 28 και 32oC) εφαρμόστηκαν στην πρώιμη οντογενετική περίοδο: για το διάστημα 0-20 dpf* (1ο πείραμα) και 10-20 dpf (2ο πείραμα). Πραγματοποιήθηκε απομόνωση ολικού RNA από τα άτομα ηλικίας 20 dpf όλων των πληθυσμών και από τα άτομα ηλικίας 10 dpf του πληθυσμού των 28oC του 2ου πειράματος και ακολούθησε υβριδοποίηση σε ολιγονουκλεοτιδικές μικροσυστοιχίες Affymetrix, με 15.509 αντιπροσωπευτικές αλληλουχίες γονιδίων (probe sets). Τα 21 μεταγραφικά προφίλ επεξεργάσθηκαν με τα εξειδικευμένα προγράμματα ανάλυσης μικροσυστοιχιών, dChip και MeV (v.4.5.1). Η κανονικοποίηση και το φιλτράρισμα των δεδομένων των μικροσυστοιχιών απέδωσε μεταγραφικά πρότυπα με 9.488 probe sets. Με τεχνικές πολυπαραμετρικής στατιστικής ανάλυσης (HCL και PCA) πραγματοποιήθηκαν οι συγκρίσεις των μεταγραφικών προτύπων μεταξύ των πειραματικών πληθυσμών για κάθε θερμοκρασία ανάπτυξης και οντογενετικό στάδιο. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων μεταξύ των δύο πειραμάτων, ii) σαφή διαχωρισμό των προτύπων των 28oC και 32oC ως προς αυτά των 22oC και στα δύο πειράματα, και iii) σαφή διαχωρισμό των προτύπων των 28oC διαφορετικού οντογενετικού σταδίου (ηλικίας 10 dpf vs 20 dpf). Θα αναμέναμε τα πρότυπα έκφρασης των 28oC να παρουσιάζουν παρόμοιο πρότυπο, κάτι που δεν παρατηρείται. Αυτό οφείλεται στο πειραματικό σφάλμα που υπεισέρχεται από το διαφορετικό χρόνο πραγματοποίησης των δύο πειραμάτων και τις ρυθμίσεις κατά την υβριδοποίηση. Έτσι πραγματοποιήθηκε η κανονικοποίηση των “28”, που απαλείφει το πειραματικό σφάλμα. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων των 28oC και 32oC ως προς αυτά των 22oC ως αποτέλεσμα της επίδρασης της θερμοκρασίας ανάπτυξης, ii) σαφή διαχωρισμό των προτύπων των 22oC των δύο πειραμάτων ως αποτέλεσμα της επίδρασης της περιόδου εφαρμογής και διάρκειας της θερμοκρασιακής αγωγής και iii) σαφή διαχωρισμό των πρότυπων των 28oC (ηλικίας 10 dpf vs 20 dpf) ως αποτέλεσμα της επίδρασης του οντογενετικού σταδίου. Ακολούθως, πραγματοποιήθηκε ανάλυση σημαντικότητας (SAM) και λειτουργική γονιδιωματική ανάλυση (λογισμικό DAVID) των στατιστικώς σημαντικών γονιδίων που διαφοροποιούν τα πρότυπα. Η ανάλυση των γονιδίων, ως προς την ιστοειδική έκφραση ανέδειξε γονίδια που σχετίζονται με την ανάπτυξη του ματιού, των θωρακικών πτερυγίων και του εγκεφάλου στους πληθυσμούς των 22oC έναντι των 28oC και 32oC. Η παρούσα εργασία αποδεικνύει την επίδραση της θερμοκρασίας ανάπτυξης και της διάρκειας της θερμοκρασιακής αγωγής, καθώς επάγει μηχανισμούς πλαστικότητας στο επίπεδο της γονιδιακής έκφρασης. Τέλος, υποδεικνύεται πως η πρώιμη οντογενετική περίοδος τείνει να είναι περισσότερο θερμοευαίσθητη, καθώς παρατηρείται εντονότερη επίδραση της θερμοκρασίας (στην περίπτωση των 22οC)στην ανάπτυξη του ατόμου. *dpf: days post fertilization
Developmental temperature plays a principal role in the ontogeny of fish. It is known that developmental temperature may shift the initiation time of the ontogenetic stages and induce plasticity in morphological and physiological characters e.g. the musculoskeletal and the cardiovascular system. However, its effect on the gene expression pattern has not previously been attempted for zebrafish. In the present study, zebrafish Affymetrix microarrays of 15,509 probe sets were used to map the transcriptome profile of: a) 20 dpf* old zebrafish larvae at three developmental temperatures, i.e. 22oC, 28oC and 32oC (1st experiment) and b) 20 dpf old zebrafish larvae, which were all grown at 28oC for the first 10 days and subsequently divided into three groups, which were grown at 22oC, 28oC and 32oC, respectively; the profile of 10 dpf old larvae was also measured (2nd experiment). We have isolated total RNA from the above populations and then, hybridization of RNA samples has been done on oligonucleotide Affymetrix microarrays of 15,509 probe sets. All 21 profiles were normalized and filtered (dChip software), and multivariate statistical analysis techniques were used on the normalized 9,488 probe set expression profiles (TM4 MeV software). Hierarchical Clustering (HCL) and Principal Component Analysis (PCA) on expression profiles indicated: a) clear separation of the two experiments based on their transcriptomic patterns, b) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC in both experiments and c) clear separation of the 28oC profiles based on the developmental stage. We would expect expression profiles of 28oC to be clustered together, though this was not observed because of experimental parameters during the hybridization, as the two experiments were carried out independently on different dates. So the normalization of “28” profiles took place, in order to eliminate the experimental noise. HCL and PCA, then, indicated: a) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC, as the effect of developmental temperature, b) clear separation of 22oC profiles of the two experiments, based on the effect of the period and duration of thermal conditions and c) clear separation of the 28oC profiles based on the developmental stage. Then, Significant Analysis of Microarrays (SAM) and Functional Genomic Classification Analysis (DAVID software) of statistically significant genes was carried out. Analysis of genes based on tissue-specific expression indicated characteristic genes for the development of the eye, pectoral fins and brain in 22oC profiles versus 28oC and 32oC profiles. The present study has proved that thermal effect is determinative among the early ontogenetic stage, especially in the case of longer cold thermal period, and developmental temperature may induce plastic response of gene expression, that could affect the fate of fish. *dpf: days post fertilization

The goal of this thesis is to algorithmically identify candidate modifiers for retinitis pigmentosa (RP) to help improve therapy and predictions for this genetic disorder that may lead to a complete loss of vision. A current research by (Chow et al., 2016) focused on the genetic contributors to RP by trying to recognize a correlation between genetic modifiers and phenotypic variation in female Drosophila melanogaster, or fruit flies. In comparison to the genome-wide association analysis carried out in Chow et al.’s research, this study proposes using a K-Means clustering algorithm on RNA expression data to better understand which genes best exhibit characteristics of the RP degenerative model. Validating this algorithm’s effectiveness in identifying suspected genes takes priority over their classification.

This study investigates the linear relationship between Drosophila eye size and genetic expression to gather statistically significant, strongly correlated genes from the clusters with abnormally high or low eye sizes. The clustering algorithm is implemented in the R scripting language, and supplemental information details the steps of this computational process. Running the mean eye size and genetic expression data of 18,140 female Drosophila genes and 171 strains through the proposed algorithm in its four variations helped identify 140 suspected candidate modifiers for retinal degeneration. Although none of the top candidate genes found in this study matched Chow’s candidates, they were all statistically significant and strongly correlated, with several showing links to RP. These results may continue to improve as more of the 140 suspected genes are annotated using identical or comparative approaches.