Browsing by Subject "microarray"
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Item A novel method for finding small highly discriminant gene sets(Texas A&M University, 2004-11-15) Gardner, Jason H.In a normal microarray classification problem there will be many genes, on the order of thousands, and few samples, on the order of tens. This necessitates a massive feature space reduction before classification can take place. While much time and effort has gone into evaluating and comparing the performance of different classifiers, less thought has been spent on the problem of efficient feature space reduction. There are in the microarray classification literature several widely used heuristic feature reduction algorithms that will indeed find small feature subsets to classify over. These methods work in a broad sense but we find that they often require too much computation, find overly large gene sets or are not properly generalizable. Therefore, we believe that a systematic study of feature reduction, as it is related to microarray classification, is in order. In this thesis we review current feature space reduction algorithms and propose a new, mixed model algorithm. This mixed-modified algorithm uses the best aspects of the filter algorithms and the best aspects of the wrapper algorithms to find very small yet highly discriminant gene sets. We also discuss methods to evaluate alternate, ambiguous gene sets. Applying our new mixed model algorithm to several published datasets we find that our new algorithm outperforms current gene finding methods.Item Arabidopsis Thaliana CARBOXYL-TERMINAL DOMAIN PHOSPHATASE-Like1 (CPL1) Mediates Responses to Iron Deficiency and Cadmium Toxicity(2014-04-24) Aksoy, EmreThe expression of genes that control iron (Fe) uptake and distribution (i.e., Fe utilization- related genes) is under a strict regulation. Fe deficiency strongly induces Fe utilization- related gene expression; however, little is known about the mechanisms that regulate this response in plants. In this dissertation, a RNA metabolism factor, RNA POLYMERASE II CTD-PHOSPHATASE-LIKE1 (CPL1) was shown to localize to the root stele, and to be involved in the regulation of Fe deficiency responses in Arabidopsis thaliana. An analysis of multiple cpl1 alleles established that cpl1 mutations enhanced transcriptional responses of Fe utilization-related genes, e.g. IRON-REGULATED TRANSPORTER1 (IRT1), to low Fe availability. In addition to the lower Fe content in the roots, but higher Fe content in the shoots of cpl1-2 plants, the root growth of cpl1-2 showed improved tolerance to Fe deficiency. Genetic data indicated that cpl1-2 likely activates Fe deficiency responses upstream of both FE?DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)- dependent and -independent signaling pathways. Interestingly, various osmotic stress/ABA-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. Unlike Fe, accumulation of the heavy-metal cadmium (Cd) in plants is toxic and it is absorbed by the roots due to the low selectivity of metal transporters such as AtIRT1. In this dissertation, CPL1 was also shown to regulate the transcriptional responses to Cd toxicity. cpl1-2 showed higher tolerance to the Cd toxicity by enhancing the root-to-shoot translocation of Cd by an unknown mechanism. A knowledge-based screening resulted in identification of a putative metal transporter, OLIGOPEPTIDE TRANSPORTER (OPT), which was highly induced in cpl1-2 upon exposure to Cd. OPT was localized to the plastids, indicating a role of plastids in Cd transport and accumulation. The root growth of opt mutants showed higher tolerance to the Cd toxicity, and the mutants accumulated less Cd, Fe and Zn, indicating the involvement of OPT in the transport of these metals. This presented dissertation suggests that 1) CPL1 functions as a negative regulator of the Fe deficiency signaling at the crosstalk with a branch of the osmotic stress/ABA signaling pathway, and 2) CPL1 regulates the Cd distribution in plants by repressing the expression of OPT.Item Control of rhythmic output from the circadian clock in Neurospora crassa(Texas A&M University, 2005-02-17) Lewis, Zachary AustinCircadian rhythms are visible as daily oscillations in biochemical, physiological, or behavioral processes. These rhythms are produced by an endogenous clock that maintains synchrony with the external environment through responses to external stimuli such as light or temperature. The clock, in turn, coordinates internal processes in a time-dependent fashion. Genetic and molecular analysis of the filamentous fungus Neurospora crassa has demonstrated that the products of the frequency (frq) and white-collar (wc-1 and wc-2) genes interact to form an interlocked feedback loop that lies at the heart of the clock in this fungus. This feedback loop, termed the FRQ/WC oscillator, produces a ~24h oscillation in frq mRNA, FRQ protein, and WC-1 protein. In turn, the FRQ/WC oscillator regulates rhythmic behavior and gene expression. The goal of this dissertation is to understand how rhythmic outputs are regulated by the FRQ/WC oscillator in Neurospora. To this end, we have taken a microarray approach to first determine the extent of clock-controlled gene expression in Neurospora. Here, we show that circadian regulation of gene expression is widespread; 145 genes, representing 20% of the genes we analyzed, are clock-controlled. We show that clockregulation is complex; clock-controlled genes peak at all phases of the circadian cycle. Furthermore, we demonstrate the clock regulates diverse biological processes, such as intermediary metabolism, translation, sexual development and asexual development. WC-1 is required for all light- and clock-regulated gene expression in Neurospora. We have shown that overexpression of WC-1 is sufficient to activate clock-controlled gene expression, but is not sufficient to induce all light-regulated genes in Neurospora. This result indicates that cycling of WC-1 is sufficient to regulate rhythmic expression of a subset of clockcontrolled genes. Conversely, a post-translational mechanism underlies WC-1 mediated light signal transduction in Neurospora. Finally, we have demonstrated the Neurospora circadian system is comprised of mutually coupled oscillators that interact to regulate output gene expression in the fungus.Item The effects of low-shear modeled microgravity on Streptococcus pneumoniae and adherent-invasive Escherichia coli(2007-07-20) Christopher Ashley Allen; Dr. Alfredo Torres; Dr. Ray Stowe III; Dr. Duane Pierson; Dr. David Niesel; Dr. Ashok ChopraThe effects of low-shear modeled microgravity (LSMMG) were investigated on Streptococcus pneumoniae global gene expression and on adherent-invasive Escherichia coli (AIEC) physiology and colonization properties. Habitation in space exposes both humans and microbes to microgravity conditions which are characterized by reductions in fluid shear forces. Areas of low-shear stress are also encountered in physiologically relevant regions of the body including the respiratory, gastrointestinal, and urogenital tracts. The LSMMG environment impacts both bacterial physiology and virulence properties and can be modeled using rotating-wall bioreactors known as high-aspect ratio vessels (HARVs). \r\nPrevious studies have evaluated the global transcriptional profiles of Gram-negative bacteria; however, no Gram-positive species have been examined. Microarray analysis of S. pneumoniae strain TIGR4 (serotype 4), after growth under LSMMG, revealed a dramatic down-shift in gene expression based on cluster analysis. Within this group of responsive genes, statistical analyses revealed that the expression of 81 genes was significantly altered. These genes were found to be associated with 7 different functional categories, including many which were uncharacterized. Several gene groups shared common functional operons and regulons such as those involved in competence induction, antimicrobial peptide production, and carbohydrate uptake. \r\nWhile previous studies examining the effects of LSMMG on bacteria have focused on well-characterized strains of both commensal and pathogenic species, there is limited information regarding the effects of LSMMG on clinical isolates associated with Crohn’s Disease, an inflammatory bowel pathology. Analysis of wild-type AIEC strain O83:H1 and an isogenic rpoS mutant (CAA001), after growth under LSMMG, revealed alterations in environmental stress resistance and increased adherence. Altered resistances to thermal and osmotic stresses were observed by LSMMG-grown AIEC O83:H1, while resistance to oxidative and acid stresses appeared to be rpoS-dependent. Further, CAA001 displayed a hyper-adherent phenotype while grown under LSMMG. TnphoA mutagenesis was used to abolish the hyper-adherent phenotype of CAA001 under LSMMG, and the insertion was mapped within the tnaB gene, encoding tryptophan permease. Complementation of the tnaB gene in the rpoS tnaB double-mutant restored adherence capabilities. These findings extend our understanding of how mechanical forces (e.g. LSMMG) can affect the functions of Gram-positive and Gram-negative species.\r\nItem Fitting It All Together: How Courtship- and Mating-Responsive Genes Affect Drosophila melanogaster Male Behavior(2011-10-21) Ellis, Lisa LynnBehavior is a complex process resulting from the integration of genetic and environmental information. Thus, the genetically tractable Drosophila melanogaster was utilized to better understand the interplay between these factors since Drosophila males and females exhibit sex-specific courtship behaviors that are innate yet modifiable. These sex-specific behaviors, as well as sexually dimorphic development, are regulated, in part, by the somatic sex-determination hierarchy. Since reproductive behaviors rely on the rapid integration of multiple sensory cues, it is likely that the perception and integration of such cues and mating-induced physiological changes are mediated in part by changes in gene expression. Therefore, it was hypothesized that assaying gene expression changes in response to courtship or mating in Drosophila males would uncover new targets of the sex-determination hierarchy and other behaviorally important loci. We took a novel approach to find these behaviorally-responsive loci by utilizing microarray technology to assess courtship- or mating-induced gene expression changes in Drosophila male whole bodies or heads. Mutations in candidate loci were tested for effects on reproductive behaviors and present the first data showing that egghead (egh) and female-specific independent of transformer (fit) affect male reproductive behavior. egh is up regulated in male heads 20 min after courting and is required post-developmentally in a subset of neurons for robust male courtship behavior. fit, a fat body-expressed sex-determination hierarchy target gene, is up regulated in male whole bodies after 5 min of courtship. fit is also up regulated in male heads after 20 min of courtship or 2 hrs after mating. Mutations in fit result in male-male courtship; more specifically, fit mutants direct courtship towards males and also elicit courtship from wild-type males. By analyzing fit's role in courtship behavior, we also shed light on the role the fat body plays in modulating behavior. These studies provide the first pieces of evidence that gene expression changes occur in Drosophila males performing reproductive behaviors. This novel approach identified behaviorally important loci that are expressed in the nervous system and the fat body, indicating that both tissues modulate behavior. Also identified were sex-determination hierarchy target genes and it is likely that further analysis of the remaining candidates will reveal more members of this genetic cascade.Item Genome-wide Transcriptome Analysis of Laminar Tissue During the Early Stages of Experimentally Induced Equine Laminitis(2012-02-14) Wang, JixinEquine laminitis is a debilitating disease that causes extreme sufferring in afflicted horses and often results in a lifetime of chronic pain. The exact sequence of pathophysiological events culminating in laminitis has not yet been characterized, and this is reflected in the lack of any consistently effective therapeutic strategy. For these reasons, we used a newly developed 21,000 element equine-specific whole-genome oligoarray to perform transcriptomic analysis on laminar tissue from horses with experimentally induced models of laminitis: carbohydrate overload (CHO), hyperinsulinaemia (HI), and oligofructose (OF). Samples were collected during the developmental (DEV) and Obel grade 1 (OG1) stages of laminitis for the CHO model. For the HI model, samples were collected at the Obel grade 2 (OG2) stage. For the OF model, samples were collected at the 12 h and 24 h time points. Appropriate control samples were obtained for all models. This is the first genome-wide transcriptome analysis of laminar tissue using an equine 21,000 70-mer long oligoarray approach in CHO, HI and OF induced laminitis. Overall, we identified the differential expression of genes encoding S100 calcium binding proteins, extracellular matrix proteins, glycoproteins, transporters, olfactory receptors, genes involved in signal transduction, body?s homeostasis, apoptosis, and immune response. Between CHO and OF models of laminitis, there were more shared genes. We discovered several common differentially expressed genes (i.e., ADAMTS1, CYCS and CXCL14) among all three models that are likely important to the pathogenesis of equine laminitis. We also discovered what appear to be central roles of apoptosis, inflammatory response, and intracellular ion homeostasis molecular processes in CHO and OF models of laminitis. Pathway analysis detected the NOD-like receptor signaling pathway, which is involved in recognition of intracellular bacteria in both the CHO and OF models of laminitis. Genetic network analysis indicated convergent pathway core molecules present in equine acute laminitis: p38 MAPK and NF-?B. Most importantly, our results of overexpression of anti-microbial genes (i.e., DEFB4, PI3, and CXCL14) suggest the central involvement of these genes in the progression of early equine laminitis and will allow refinement of current hypotheses of disease pathogenesis.Item Genomic analyses of induced hypercholesterolemia and atherosclerosis in a mixed breed colony of dogs and developmental abnormalities in the Havanese(2009-05-15) Starr, Alison NicoleThe domestic dog, Canis lupus familiaris, is a unique model system for the dissection of hereditary diseases. Selective breeding practices have created more than 300 distinct breeds of dogs, born from a desire to create specific physical and behavioral characteristics. Breeds represent closed breeding populations and the extensive records maintained for members of each breed (e.g., multi-generational pedigrees, veterinary medical records) present an incredible tool for genetic research. Two closed populations were used in the work presented here: a colony of mixed-breed dogs segregating resistance and sensitivity to cholesterol feeding, and a purebred pet population of Havanese experiencing a high frequency of developmental abnormalities. Estimates of heritability were calculated for each disease to evaluate the degree of phenotypic variation attributable to genetics among dogs in the populations used. A heritability of 0.55 (? 0.16) was identified for cholesterol resistance and sensitivity in the mixed-breed colony. The small sample size prevented the use of complex segregation analyses to examine mode of transmission. A heritability of 0.36 (? 0.26) was calculated for the composite phenotype in the Havanese, encompassing the spectrum of abnormalities in the breed. Polygenic inheritance was identified for the composite phenotype, but the action of a major gene was identified by complex segregation analyses in the Havanese. Complex diseases preclude the use of a candidate gene approach, owing to the multitude of genes involved in the disease process. Whole genome screens provide a practical approach to the identification of chromosomal region(s) associated with a disease phenotype by narrowing the search for candidate gene(s). The Minimal Screening Set ? 2 (MSS-2) was used in the present studies to evaluate the segregation of microsatellite markers in pedigrees for both the mixed-breed colony and the Havanese. No significant LOD scores were identified, though suggestive LOD scores were obtained in both analyses. A canine-specific oligonucleotide microarray was used to create gene expression profiles for developmental abnormalities in the Havanese and for cholesterol sensitivity in the mixed-breed colony dogs. Distinct expression profiles were generated for each group, and several genes of interest were identified as being both differentially expressed (>?2-fold change) and statistically significant (p-value<0.05).Item Genomic Approaches to Study Innate Immune Response to Salmonella Enteritidis Infection in Chickens(2010-01-14) Chiang, Hsin-ISalmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis. Contamination of consumed poultry products continues to be a global threat to public health. Genetic resistance using genomic approach provides a promising solution to controlling SE infection in poultry. The mechanism of SE resistance in chickens remains elusive. Three different approaches, microarray techology, gene silencing, and computational gene analysis, have been utilized to study SE-induced transcriptional changes of host immune response in the chicken. A whole genome chicken 44K microarray was used to analyze the transcriptome of heterophils from SE-resistant (line A) and SE-susceptible chickens (line B) with/without in vitro SE stimulation. Many differentially expressed immune-related genes were found in the SE-infected to non-infected comparison, where more immune-related genes were down-regulated in line B than line A. These results suggested a similar Toll-like receptor (TLR) regulatory network might exist in heterophils of both lines, and provided strong candidates for further investigating SE resistance and susceptibility in chickens. In the gene silencing study, small interfering RNAs (siRNA) were used to specifically inhibit the expression of NFkB1 in the chicken HD11 macrophage cell line with SE challenge. Genes related to the NF-kB signaling pathway were selected to examine the effect of NFkB1 inhibition on TLR pathway. With 36% inhibition of NFkB1 expression, the results showed an increased expression of TLR4 and interleukin (IL)-6 following SE challenge and suggested a likely inhibitory regulation of NFkB1 on TLR signal pathway. Finally, two novel chicken C-type lectin-like receptors were identified and annotated to chicken CD69 and CD94/NKG2-like with multiple evidences generated by computational (in-silico) sequence analysis. Both genes located in a region on chicken chromosome 1 that is syntenic to mammalian Nature Killer Receptor Complex (NKC) region, which may have existed before the divergence between mammals and aves. While siRNA lays the foundation of using loss-of-function approach on testifying gene-gene interactions, in-silico analysis aids in gathering information of unknown genes of great interest. Both approaches provide great potential to use for down-stream analysis following microarray study.Item Identification of Significantly Regulated Genes in the Estrogen Induced Gallus gallus Liver Over a 24-Hour Time Course(2012-02-14) Trojacek, EricaIn birds, estrogen is a strong stimulator of gene programs that regulate the formation of very low density lipoproteins (VLDL). Apolipoprotein-B (ApoB) is an integral part of very low density lipoproteins. In mammals, the rate of ApoB synthesis is controlled by post-translational means. In contrast, estrogen treated birds show changes in ApoB transcript level; in a natural setting, the bird?s metabolism and transcription are in great flux due to yolk formation. Besides the ApoB gene, the entire complement of genes that is necessary to form a VLDL is not known. To determine the genes that play a role in the formation of VLDL 7-10d old chicks were injected with estrogen at several time points over a 24hr period. Following exsanguinations by cardiac puncture, livers were removed and RNA was extracted. The RNA was quantified and hybridized to microarrays using a dual-dye system. Slides were scanned and analyzed, and features were extracted. To qualify microarray results, quantitative real time PCR (q-RTPCR) was done on a selection of genes. Previous studies had shown that approximately 200 genes are upregulated by the treatment of hormone naive chickens with estrogen. As a result of our liver transcriptional profiling, we identified 1,528 genes at 1.5hrs, 1,931 genes at 3hrs, 2,398 genes at 6hrs, 2,356 at 12hrs, and 1,713 genes at 24hrs following estrogen exposure. We determined that these regulated genes include those responsible for the transcription of RNA used to create the gene products that serve as components of VLDL itself or that act in VLDL assembly. These include genes encoding structural proteins, like ApoB, and genes encoding assembly-related proteins. Of the differentially expressed genes as compared to time 0, there were approximately 30% which were unannotated with regards to function limiting conclusions. We hope to determine the function of these genes and to annotate them based on this information.Item Implementation of genomics and bioinformatics approaches for identification and characterization of tomato ripening-related genes(Texas A&M University, 2004-09-30) Fei, ZhangjunInitial activities were focused on isolation and characterization of fruit ripening-related genes from tomato. Screening of four tomato cDNA libraries at low stringency with 10 fruit development and ripening-related genes yielded ~3000 positives clones. Microarray expression analysis of half of these positives in mature green and breaker stage fruits resulted in eight ripening-induced genes. RNA gel-blot analysis and previously published data confirmed expression for seven of the eight. One novel gene, designated LeEREBP1, was chosen for further characterization. LeEREBP1 encodes an AP2/ERF-domain transcription factor and is ethylene inducible. The expression profiles of LeEREBP1 parallel previously characterized ripening-related genes from tomato. Transgenic plants with increased and decreased expression of LeEREBP1 were generated and are currently being characterized to define the function of LeEREBP1. A large public tomato EST dataset was mined to gain insight into the tomato transcriptome. By clustering genes according to the respective expression profiles of individual tissues, tissue and developmental expression patterns were generated and genes with similar functions grouped together. Tissues effectively clustered for relatedness according to their profiles confirming the integrity of the approach used to calculate gene expression. Statistical analysis of EST prevalence in fruit and pathogenesis-related libraries resulted in 333 genes being classified as fruit ripening-induced, 185 as fruit ripening-repressed, and 169 as pathogenesis-related. We performed a parallel analysis on public EST data for grape and compared the results for ripening-induced genes to tomato to identify similar and distinct ripening factors in addition to candidates for conserved regulators of fruit ripening. An online interactive database for tomato gene expression data - Tomato Expression Database (TED) was implemented. TED contains normalized expression data for approximately 12,000 ESTs over ten time points during fruit development. It also contains comprehensive annotation of each EST. Through TED, we provide multiple approaches to pursue analysis of specific genes of interest and/or access the larger microarray dataset to identify sets of genes that may behave in a pattern of interest. In addition, a set of useful data mining and data visualization tools were developed and are under continuing expansion.Item Integrative analysis of high-throughput biological data: shrinkage correlation coefficient and comparative expression analysis(2009-12) Yao, Jianchao; Roux, Stanley J.; Chen, Zengjian J.; Markey, Mia K.; Miranker, Daniel P.; Fu, BoThe focus for this research is to develop and apply statistical methods to analyze and interpret high-throughput biological data. We developed a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. This computational approach is not only applicable to DNA microarray analysis but is also applicable to proteomics data or any other high-throughput analysis methodology. The suppression of APY1 and APY2 in mutants expressing an inducible RNAi system resulted in plants with a dwarf phenotype and disrupted auxin distribution, and we used these mutants to discover what genes changed expression during growth suppression. We evaluated the gene expression changes of apyrase-suppressed RNAi mutants that had been grown in the light and in the darkness, using the NimbleGen Arabidopsis thaliana 4-Plex microarray, respectively. We compared the two sets of large-scale expression data and identified genes whose expression significantly changed after apyrase suppression in light and darkness, respectively. Our results allowed us to highlight some of the genes likely to play major roles in mediating the growth changes that happen when plants drastically reduce their production of APY1 and APY2, some more associated with growth promotion and others, such as stress-induced genes, more associated with growth inhibition. There is a strong rationale for ranking all these genes as prime candidates for mediating the inhibitory growth effects of suppressing apyrase expression, thus the NimbleGen data will serve as a catalyst and valuable guide to the subsequent physiological and molecular experiments that will be needed to clarify the network of gene expression changes that accompany growth inhibition.Item Land use and land cover change: the effects of woody plant encroachment and prescribed fire on biodiversity and ecosystem carbon dynamics in a southern great plains mixed grass savanna(2009-05-15) Hollister, Emily BrookeIn the southern Great Plains, the encroachment of grassland ecosystems by mesquite (Prosopis glandulosa), is widespread, and prescribed fire is commonly used in its control. Despite this, substantial quantitative information concerning their influences on the community composition, functional dynamics, and soil organic carbon (SOC) storage potential of grassland ecosystems is lacking. The objectives of this study were to: a) quantify the effects of seasonal prescribed fire treatments and mesquite encroachment on aboveground net primary productivity (ANPP) and herbaceous community composition; b) characterize SOC pool sizes, turnover, and storage potential relative to vegetation type and fire treatment; c) evaluate the structure and diversity of soil microbial communities relative to vegetation type; and d) characterize the functional diversity of these same microbes using the GeoChip functional gene microarray. Repeated winter and summer fires led to increased ANPP rates (average, 434 and 313 g m-2 y-1, respectively), relative to unburned controls (average, 238 g m-2 y-1), altered herbaceous community composition, and increased the storage of resistant forms of SOC, but did not affect overall SOC storage. Herbaceous ANPP rates did not differ significantly as a result of mesquite encroachment, but herbaceous community composition and SOC storage did. Mesquite soils contained significantly more total, slow-turnover, and resistant forms of SOC than those that occurred beneath C3 or C4 grasses. Similarity among the soil bacterial and fungal communities associated with the major vegetation types in this system was low to moderate. Significant differences were detected among soil fungi, with the mesquite-associated fungi harboring significant differences in community structure relative to the fungal communities associated with each of the other vegetation types examined. Despite this result, few significant differences were detected with respect to the functional diversity of these communities, suggesting either a high degree of functional redundancy, or that the functional differences harbored by these communities are beyond the scope of the GeoChip. The results of this study demonstrate that both fire and mesquite encroachment have the potential to alter ecosystem components and processes significantly, providing new insight regarding the effects of these widespread land use and land cover changes on ecosystem structure and function.Item Regulation of Branching by Phytochrome and Phytohormones(2012-07-16) Krishnareddy, Srirama R.Light is the fundamental source of energy and information throughout the plant life cycle. Light signals regulate plant architecture and branching, key processes that determine biomass production and grain yield. Low red (R) to far-red (FR) light ratios (R:FR) perceived by phytochromes serve as a warning signal about impending competition for light resources and lead to shade avoidance responses (SARs), including reduced branching. The R:FR regulates branching in both a bud autonomous and non-bud autonomous manner, however a detailed mechanistic understanding of the process remains unclear. We hypothesized that high R:FR promotes bud outgrowth by differentially regulating branching-related genes (transcriptome) within the axillary bud and that increased apical dominance under low R:FR or with phyB deficiency is mediated by auxin or other novel signal/s. We analyzed the branching phenotype of Arabidopsis Columbia-60000 ecotype in response to different R:FR treatments and conducted a microarray study to identify early (within 3 hours) changes in the transcriptome of buds from different rosette positions in response to altered R:FR. Physiological experiments were also conducted to determine if auxin concentration, transport rate, sensitivity, and establishment of an auxin transport stream were important in determining the branching phenotype of shade avoiding plants. The results revealed that the duration of low R:FR determines plant architecture and the branching phenotype and that bud outgrowth is regulated by the R:FR in a spatial and temporal manner. Low R:FR promoted the elongation of branches at top rosette nodes while it suppressed the outgrowth of axillary buds at lower nodes. High R:FR could reverse the effects of previous low R:FR by promoting the outgrowth of buds from lower axils within 24 hours of treatment. Transcriptomic analysis revealed that the R:FR differentially regulated the expression of genes related to hormone biosynthesis/transport/signaling, cell-cycle regulation and cell wall modification. Cis-elements responsive to light and hormone signaling pathways were overrepresented in several gene clusters. Apical dominance related studies discovered that loss of phyB function results in a slower auxin transport rate, fewer xylem parenchyma cells, and reduced sensitivity to auxin. These results, in addition to estimates of correlative inhibition, suggested that auxin is at least partially responsible for increased apical dominance under low R:FR or with phyB deficiency, but may be acting in conjunction with other undefined regulators.Item Roles for extra-hypothalamic oscillators in the avian clock(2009-05-15) Karaganis, Stephen PaulAvian circadian clocks are composed of a distributed network of neural and peripheral oscillators. Three neural pacemakers, located in the pineal, the eyes, and the hypothalamus, control circadian rhythms of many biological processes through complex interactions with slave oscillators located throughout the body. This system, an astonishing reflection of the life history of this diverse class of vertebrates, allows birds to coordinate biochemical and physiological processes and harmonize them with a dynamic environment. Much work has been done to understand what roles these pacemakers have in avian biology, how they function, and how they interact to generate overt circadian rhythms. The experimental work presented in this dissertation uses the domestic chicken, Gallus domesticus, as a model to address these questions and carry forward current understanding about circadian biology in this species. To do so, we utilized a custom DNA microarray to investigate rhythmic transcription in cultured chick pineal cells. We then sought to identify genes which might be a component of the pineal clock by screening for rhythmic transcripts that are sensitive to a phase-shifting light stimulus. Finally, we surgically removed the eyes or pineal from chickens to examine the roles of these extra-SCN pacemakers in regulating central and peripheral rhythms in metabolism and clock gene expression. Using these methods, we show that the oscillating transcriptome is diminished in the chick pineal ex vivo, while the functional clustering of clock controlled genes is similar. This distribution reveals multiple conserved circadian regulated pathways, and supports an endogenous role for the pineal as an immune organ. Moreover, the robustness of rhythmic melatonin biosysnthesis is maintained in vitro, demonstrating that a functional circadian clock is preserved in the reduced subset of the rhythmic pineal transcriptome. In addition, our genomic screen has yielded a list of 28 genes that are candidates for functional screening. These should be evaluated to determine any potential role they may have as a component of the pineal circadian clock. Finally, we report that the eyes and pineal similarly function to reinforce rhythms in brain and peripheral tissue, but that metabolism and clock gene expression are differentially regulated in chick.Item Small sample feature selection(Texas A&M University, 2007-09-17) Sima, ChaoHigh-throughput technologies for rapid measurement of vast numbers of biolog- ical variables offer the potential for highly discriminatory diagnosis and prognosis; however, high dimensionality together with small samples creates the need for fea- ture selection, while at the same time making feature-selection algorithms less reliable. Feature selection is required to avoid overfitting, and the combinatorial nature of the problem demands a suboptimal feature-selection algorithm. In this dissertation, we have found that feature selection is problematic in small- sample settings via three different approaches. First we examined the feature-ranking performance of several kinds of error estimators for different classification rules, by considering all feature subsets and using 2 measures of performance. The results show that their ranking is strongly affected by inaccurate error estimation. Secondly, since enumerating all feature subsets is computationally impossible in practice, a suboptimal feature-selection algorithm is often employed to find from a large set of potential features a small subset with which to classify the samples. If error estimation is required for a feature-selection algorithm, then the impact of error estimation can be greater than the choice of algorithm. Lastly, we took a regression approach by comparing the classification errors for the optimal feature sets and the errors for the feature sets found by feature-selection algorithms. Our study shows that it is unlikely that feature selection will yield a feature set whose error is close to that of the optimal feature set, and the inability to find a good feature set should not lead to the conclusion that good feature sets do not exist.Item Small sample multiple testing with application to cDNA microarray data(Texas A&M University, 2006-10-30) Hintze, Eric PooleMany tests have been developed for comparing means in a two-sample scenario. Microarray experiments lead to thousands of such comparisons in a single study. Several multiple testing procedures are available to control experiment-wise error or the false discovery rate. In this dissertation, individual two-sample tests are compared based on accuracy, correctness, and power. Four multiple testing procedures are compared via simulation, based on data from the lab of Dr. Rajesh Miranda. The effect of sample size on power is also carefully examined. The two sample t-test followed by the Benjamini and Hochberg (1995) false discovery rate controlling procedure result in the highest power.Item Sorghum gene expression modulated by water deficit and cold stress(Texas A&M University, 2007-04-25) Lim, SanghyunGlobal gene expression in Sorghum bicolor, an important crop showing drought tolerance in arid and semi-arid cultivated areas, was monitored to exposure of 8-days seedlings to water deficit (20% polyethylene glycol) or cold stress (4 ????C). A sorghum cDNA microarray, including ~13,000 (milestone version 1) or ~28,000 (milestone version 2) unigenes, was used to examine gene expression in shoots and roots at 3 and 27hours after stress treatment. ~1,300 and ~2,300 genes were modulated by water deficit and cold stress, respectively. Up-regulated genes included previously identified stressinduced genes such as early drought-induced gene, dehydrin, late embryogenesis abundant gene, glycin and proline-rich gene, and water stress-inducible genes as well as unknown genes. Genes involved in signal transduction, lipid metabolism, transporter, and carbohydrate metabolism are induced. Quantitative real-time PCR was used to quantify changes in relative mRNA abundance for 333 and 108 genes in response to water deficit and cold stress, respectively. Stress-induced genes were classified by kinetics. Eighteen of 108 cold-induced genes were modulated by cold but not by ABA and PEG treatment. This research provides the starting point for detailed analysis and comparison of water deficit and cold modulated gene networks in sorghum.Item Topics in genomic image processing(Texas A&M University, 2006-04-12) Hua, JianpingThe image processing methodologies that have been actively studied and developed now play a very significant role in the flourishing biotechnology research. This work studies, develops and implements several image processing techniques for M-FISH and cDNA microarray images. In particular, we focus on three important areas: M-FISH image compression, microarray image processing and expression-based classification. Two schemes, embedded M-FISH image coding (EMIC) and Microarray BASICA: Background Adjustment, Segmentation, Image Compression and Analysis, have been introduced for M-FISH image compression and microarray image processing, respectively. In the expression-based classification area, we investigate the relationship between optimal number of features and sample size, either analytically or through simulation, for various classifiers.Item Transcript profiling of differentiating xylem of loblolly pine (Pinus taeda L.)(Texas A&M University, 2005-02-17) Yang, Suk-HwanWood formation (xylogenesis) is a critical developmental process for all woody land plants. As an initial step to understand the molecular basis for temporal and spatial regulation of xylogenesis and the effect of the expression of individual genes on physical and chemical properties of wood, microarray and realtime RTPCR analyses were performed to monitor gene expression during xylogenesis under various developmental and environmental conditions. The specific objectives established for this study were: Objective 1. Microarray analysis of genes preferentially expressed in differentiating xylem compared to other tissues of loblolly pine (see Chapter II); Objective 2. Microarray analysis of seasonal variation in gene expression for loblolly pines (Pinus taeda L.) from different geographical sources (see Chapter III); Objective 3. Realtime RTPCR analysis of loblolly pine AGP and AGPlike genes (see Chapter IV). Based on the results from this study, candidate genes may be further studied for association with significant traits, used for genetic modification of wood properties, or included in future studies to further examine the molecular mechanisms of wood formation.Item Wide-cross whole-genome radiation hybrid (WWRH) mapping and identification of cold-responsive genes using oligo-gene microarray analysis in cotton(Texas A&M University, 2005-02-17) Gao, WenxiangThe first part of this research focused on wide-cross whole-genome radiation hybrid (WWRH) mapping of the cotton (Gossypium) genome. Radiation hybrid mapping has been used extensively to map the genomes of human and certain animal species, but not plant species. In lieu of in vitro hybrid cell line technologies for plants, we developed a novel approach for radiation hybrid mapping based on wide-cross in vivo hybridization. Flowers from one species of cotton, either G. hirsutum or G. barbadense, were -irradiated and then used to pollinate the other species. The resulting hybrid plants were assessed as a mapping tool. Two WWRH mapping panels were constructed from 5- and 8-krad -irradiation treatments. Both panels demonstrated that the WWRH mapping method can be used to map the cotton genome, and that this method complements traditional linkage mapping approaches. The second part of this research focused on the identification of cold-responsive genes using spotted oligo-gene microarray analysis. Increased cold-tolerance in cotton would promote early and uniform seedling establishment, expand the growing season, decrease susceptibility to fungal infections and certain diseases, and increase fiber yield and quality. BLAST searches of the cotton database using amino acid sequences of 93 drought/cold-related genes from Arabidopsis and several other plant species led to 806 cotton orthologous cDNAs and expressed sequence tags (ESTs). Eight hundred and six cotton 70-mer oligos were designed and included in an oligo-gene microarray containing 1,536 70-mer oligos, each representing a cDNA or EST from cotton, or one of 121 chloroplast genes or 66 mitochondrial genes from Arabidopsis. Thirty-eight cotton cDNAs and ESTs were identified as cold-responsive genes based on experimental treatment and oligo-gene microarray analysis. Expression was up-regulated for 36 genes and down-regulated for two genes by cold treatment. Results from microarray analysis were tested and confirmed by northern blot analysis for 16 genes. Our data suggest that Arabidopsis orthologous genes can be used to identify homologous cotton genes. The oligo-gene microarray is a valid approach to study transcriptional changes in cotton.