Browsing by Subject "gene expression"
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Item Biological and functional consequences of polymorphisms in the XPD gene(2006-10-23) Kevin James Wolfe; Sherif Z.Abdel-Rahman, Ph.D.; Thomas E. Albrecht, Ph.D.; Rodney S. Nairn, Ph.D.; Randa A. El-Zein, MD. Ph.D.; Mary Treinen-Moslen, Ph.D.; Jonathan B. Ward, Ph.D.Epidemiological studies have documented many associations between single nucleotide polymorphisms (SNPs) in the nucleotide excision repair gene XPD (ERCC2) and cancer risk. Little is known, however, about the underlying mechanisms for these associations. I used lymphocytes from healthy subjects to explore novel mechanisms which could explain the reported risk-modifying effects on disease susceptibility of three SNPs in the XPD gene, the synonymous C156A SNP in exon 6 and the nonsynonymous SNPs Asp312Asn in exon 10 and Lys751Gln in exon 23. Baseline and NNK-induced chromosomal aberrations (Ca) were assessed by cytogenetic analysis and then P values were calculated as estimates of sub-population differences for increased frequencies of CAs associated with the three XPD polymorphisms. I found significant elevation in baseline frequencies of CAs among smokers with the variant 312Asn polymorphism (P=0.028). Elevations in NNK-induced aberrations were found among younger subjects (age<39 years) with the 156A or the 751Gln polymorphism, (P=0.025 and P=0.037, respectively) and in females compared to males with a combination of the 312Asn and the 751Gln polymorphisms (P=0.045). Application of real time PCR showed that each SNP, alone and in combination, significantly decreased constitutive XPD mRNA levels (P<0.003) in lymphocytes. Decreases in XPD mRNA levels were significantly higher in older subjects and in smokers. Localized Mfold structure analysis of the mRNA sequence surrounding the studied SNPs were predicted to alter mRNA secondary structure, which indicated that these SNPs potentially affect local folding and mRNA stability. UVC treatment of cells with wild type XPD produced a significant increase (P=0.03) in XPD protein levels by 30 min, which surprisingly coincided with a decrease in XPD mRNA transcript copy number (P=0.0002). Fluorescent confocal microscopy demonstrated that this increase in XPD protein appeared largely due to an increase in nuclear localization of XPD, which was evident at 30 min and persistent at 6 hrs. New observations from this project provide possible mechanistic explanations for the association of polymorphisms in XPD with increased genetic damage (CAs) and cancer risk.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 Copy Number and Gene Expression: Stochastic Modeling and Therapeutic Application(2013-05-01) Hsu, Fang-HanThe advances of high-throughput technologies, such as next-generation sequencing and microarrays, have rapidly improved the accessibility of molecular pro?les in tumor samples. However, due to the immaturity of relevant theories, analyzing these data and systematically understanding the underlying mechanisms causing diseases, which are essential in the development of therapeutic applications, remain challenging. This dissertation attempts to clarify the e?ects of DNA copy number alterations (CNAs), which are known to be common mutations in genetic diseases, on steady- state gene expression values, time-course expression activities, and the e?ectiveness of targeted therapy. Assuming DNA copies operate as independent subsystems producing gene transcripts, queueing theory is applied to model the stochastic processes representing the arrival of transcription factors (TFs) and the departure of mRNA. The copy-number-gene-expression relationships are shown to be generally nonlinear. Based on the mRNA production rates of two transcription models, one corresponding to an unlimited state with proli?c production and one corresponding to a restrictive state with limited production, the dynamic e?ects of CNAs on gene expression are analyzed. Simulations reveal that CNAs can alter the amplitudes of transcriptional bursting and transcriptional oscillation, suggesting the capability of CNAs to interfere with the regulatory signaling mechanism. With this ?nding, a string-structured Bayesian network that models a signaling pathway and incorporates the interference due to CNAs is proposed. Using mathematical induction, the upstream and downstream CNAs are found to have equal in?uence on drug e?ectiveness. Scoring functions for the detection of unfavorable CNAs in targeted therapy are consequently proposed. Rigorous experiments are keys to unraveling the etiology of genetic diseases such as cancer, and the proposed models can be applied to provide theory-supporting hypotheses for experimental design.Item Detecting Aflatoxicosis in Broilers in the Evaluation of Clay-based, Toxin-binding Feed Additives(2014-11-17) Fowler, Justin CaseThe objectives of this research were to evaluate common biological measures of aflatoxicosis in broilers (such as growth rate and relative organ weights) along with variables such as hepatic gene expression and aflatoxin residues in the liver, pursuant to identifying a more sensitive biological assay that will allow researchers to conduct three-week broiler trials at aflatoxin concentrations <1000 ppb, prior to significant changes in the growth rate or relative organ weights. This will help us both better understand how aflatoxicosis presents in broilers, as well as help us evaluate the efficacy of clay-based binders for their ability to ameliorate aflatoxicosis under experimental conditions. In the first study, a recently mined calcium bentonite clay (TX4) was evaluated against Novasil?. Both clays appeared able to sequester aflatoxin, and overall TX4 appeared capable of ameliorating aflatoxicosis comparable to Novasil?. In the second study, growth and relative organ weight data were compared with the gene expression of hepatic enzymes known to detoxify aflatoxin B1 in broilers that had consumed a wide range of aflatoxin concentrations. When gene expression data from liver samples were analyzed, the genotypic effect of aflatoxin on the CYP1A1 and CYP2H1 isoforms simply mirrored the phenotypic effects seen in the growth and relative organ weights, suggesting that this variable was not any more sensitive than the more traditional ones. The third study evaluated the TX4 clay when in diets containing <1000 ppb aflatoxin. Although weight gain was unaffected by aflatoxin at these lower levels (after three weeks on treatment diets, body weights between the 0 ppb treatment and the 700 ppb treatment only varied by 4%), there were negative effects on feed conversion and productivity index and there was an increase in the relative weights of the liver and kidney. The inclusion of TX4 to the treatment diets did not offer any amelioration from the main effects of aflatoxin. Finally, a study was conducted to evaluate the effects of TX4 clay when using residues of aflatoxin B1 in the liver as the primary variable of interest. Results after one week on treatment diets showed that TX4 was effective at reducing the accumulation of aflatoxin B1 residues in liver. However, after the first week, liver residue data were not any more sensitive in evaluating aflatoxin or clay effects when compared to the ?traditional? measures of growth performance and organ weights. Also, these results indicate that the clearance time required to remove aflatoxin residues from the liver is less than one week on a clean corn diet. Based on these evaluations, attempts to characterize a more sensitive, sentinel-type response to aflatoxin exposure in broilers were not any more successful at evaluating aflatoxicosis than was the common bioassay measures such as growth rate and relative organ weights. These studies (by contaminating corn with aflatoxigenic species of Aspergillus) were able to find significant main effects for aflatoxin at lower concentrations (?1000 ppb) than had been previously reported by the studies that included inoculated rice.Item Effects of Fatty Acids on Gene Expression and Lipid Metabolism in Bovine Intramuscular and Subcutaneous Adipose Tissues(2012-10-19) Silvey, David TyronePasture feeding depresses adipose tissue development in beef cattle whereas grain feeding, enhances adipogenesis. Therefore, we hypothesized that specific fatty acids would differentially affect lipogenesis in explants of bovine subcutaneous (SC) and intramuscular (IM) adipose tissues. Angus steers were harvested at 12, 14, and 16 mo of age, and IM and SC adipose tissue explants from the 8-11th thoracic rib region were dissected and cultured in media. Media contained no supplemental fatty acids or 40 microM of five fatty acids, stearic acid (18:0), oleic acid (18:1 n-9), trans-11 vaccenic acid (18:1 trans-11), conjugated linoleic acid (CLA, 18:2 trans-10, cis-12), or alpha-linolenic acid (18:3 n-3). After 48 h of culture, lipogenesis using [U-14C]glucose and [1-14C]acetate was measured. Lipogenesis from glucose decreased between 12 and 16 mo of age in SC adipose tissue (from 8.9 to 4.0 nmol per 2 h per 100 mg; P = 0.001) and IM adipose tissue (from 4.4 to 2.7 nmol per 2 h 100 mg ; P = 0.08). Lipogenesis from acetate did not change over time in SC adipose (approximately 56 nmol per 2 h per 100 mg; P = 0.23), but increased over time in IM adipose tissue (from from 11.3 to 17.1 nmol per 2 h 100 mg; P = 0.02). Oleic acid increased lipid synthesis from glucose 125 percent (P = 0.04) in IM adipose tissue, whereas stearic acid and trans-vaccenic acid increased lipogenesis from glucose in SC adipose tissue by approximately 50 percent (P = 0.04). In SC adipose tissue only, trans-vaccenic and increased, lipogenesis from glucose (P < 0.02). Lipogenesis from acetate was depressed by CLA nearly 50 percent in SC adipose tissue. PPAR? gene expression increased between 14 and 16 mo of age in control IM and SC adipocytes. The increase in activity was also observed in AMPK gene expression. C/EBP? and SCD gene expression did not increase in control samples until 16 mo of age. SC adipose tissue responded to stearic acid by increased GPR43 and AMPK gene expression at 12 mo of age. We conclude that fatty acids differentially affect lipid synthesis in IM and SC adipose tissues, which may account for the effects of pasture and grain feeding on adiposity.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 Elucidating the Functions of the Sialylation Pathway in Drosophila melanogaster(2012-10-19) Carnahan, MindySialylation is an important carbohydrate modification of glycoconjugates, which introduces sialic acids (SA). The relatively large nine-carbon, negatively charged sugars are typically located at the termini of carbohydrate chains. SA's are often required for functionally important molecular and cellular interactions including virus-host interactions, tumor progression and malignancy, immune system development and function, and nervous system development and function. However, the study of sialylation in vertebrates, including man, encounters serious obstacles associated with the complexity of vertebrates' biology and limitations of available experimental approaches. Drosophila is a useful model system with many advantages including quick generation time, a large number of progeny, simplified glycosylation and neurophysiology, and ease of genetic manipulations. The primary focus of this thesis is on the functions of Drosophila melanogaster CMP sialic acid synthetase (DmCSAS) and sialyltransferase (DSiaT) in the central nervous system (CNS). A combination of genetic, immunostaining, and neurobiology approaches were used to characterize the functions of DmCSAS and DSiaT in Drosophila. This investigation revealed the expression of DmCSAS and suggested that it plays an important role in a specialized and developmentally regulated process in the nervous system of Drosophila. Further experiments examined sub-cellular localization of DmCSAS revealing that this protein has a complex mostly Golgi-associated distribution within the cell in vivo. I discovered a novel link between Drosophila sialylation and circadian rhythm regulation. I also characterized the electrophysiological phenotypes of DmCSAS mutants and compared them to the corresponding defects associated with DSiaT mutations. My experiments also revealed that the relationship between DmCSAS and DSiaT are more complex than originally thought; these genes may have independent functions while also participating in the same pathway. Taken together, these results elucidate the sialylation pathway in Drosophila and shed more light on the role of sialylation in the nervous system. My experiments provide a unique evolutionary perspective on the sialylation pathway in animals and suggest that the neural function of SA in Drosophila can be conserved in vertebrates, including humans.Item Evaluation of alterations in gene expression in MCF-7 cells induced by the agricultural chemicals Enable and Diazinon(Texas A&M University, 2005-08-29) Mankame, Tanmayi PradeepSteroid hormones, such as estrogen, are produced in one tissue and carried through the blood stream to target tissues in which they bind to highly specific nuclear receptors and trigger changes in gene expression and metabolism. Industrial chemicals, such as bisphenol A and many agricultural chemicals, including permethrin and fervalerate, are known to have estrogenic potential and therefore are estrogen mimics. Widely used agricultural chemicals, Enable (fungicide) and Diazinon (insecticide), were evaluated to examine their toxicity and estrogenicity. MCF-7 cells, an estrogen-dependent human breast cancer line, were utilized for this purpose. MCF-7 cells were treated with 0.033-3.3 ppb (ng/ml) of Enable and 0.3-67 ppm of Diazinon and gene expression was compared to that in untreated cells. Microarray analysis showed down-regulation of eight genes and up-regulation of thirty four genes in cells treated with 3.3 ppb of Enable, compared to untreated cells. Similarly, in cells treated with 67 ppm of Diazinon, there were three genes down-regulated and twenty seven genes up-regulated. For both chemicals, specific genes were selected for special consideration. RT-PCR confirmed results obtained from analysis of the microarray. These studies were designed to provide base-line data on gene expression-altering capacity of specific chemicals and will allow assessment of the deleterious effects caused by exposure to the aforementioned chemicals.Item Gene Expression and Association Analyses of Stress Responses in Loblolly Pine (Pinus taeda L.)(2012-02-14) Seeve, Candace MarieThe molecular mechanisms underlying disease-resistance and drought-resistance in forest trees are not well understood. Linking variation in gene expression with genetic polymorphisms and with variations in disease- and drought-resistance phenotypes can provide information about these complex traits. We used real-time quantitative polymerase chain reaction (PCR) to detect variations in the expression of 88 disease- and drought-responsive genes within an association population of 354 loblolly pine trees (Pinus taeda L.). Using association genetics approaches, we then linked 3,938 single nucleotide polymorphisms (SNPs) in candidate genes with gene expression phenotypes to identify novel disease- and drought-responsive genes. To further examine differences in gene expression induced by drought, Fusarium circinatum (responsible for pitch canker disease), and drought F. circinatum, the expression of 114 genes identified through comparative and association genetics approaches was analyzed on a subset of 24 loblolly pine trees possessing a range of pitch canker- and drought-resistance phenotypes. Significant differences in the uninduced expression of all 88 genes measured on the association population were observed among loblolly pine trees. Principal component analysis showed that some variation within the association population could be accounted for by population substructure of geographic origin. Hierarchical clustering of genes based on uninduced expression did not consistently group together functionally similar genes probably because expression was collected on unstressed stem tissue. This was supported in the smaller expression study as correlations between expression values of genes in the same functional networks were usually stronger when induced by a treatment compared with correlations between the uninduced expression of genes in the control group. Gene expression frequently changed by up to 4-fold in response to one or more treatments, but PtMYB12 was the only gene that exhibited a statistically significant change in response to treatments. ANOVA analyses of gene expression controlling for pitch canker resistance and for water use efficiency phenotypes identified differentially expressed genes suggesting that they may be contributing to these phenotypes. Finally, association genetics approaches detected 101 significant associations between SNPs in 94 candidate genes potentially involved in stress responses and 27 gene expression phenotypes.Item Identification of Pseudomonas aeruginosa via a poplar tree model(2009-05-15) Attila, CanDifferential gene expression of P. aeruginosa in a rhizosphere biofilm on poplar tree roots was examined in order to identify new virulence factors from this human pathogen. Changes in gene expression for poplar trees contacted with P. aeruginosa was examined as well to identify the response of poplar roots to P. aeruginosa infection. This is the first study of the whole-transcriptome analysis of P. aeruginosa on a plant tree root. The 20 most highly-induced genes of P. aeruginosa were examined for their role in biofilm formation, rhizosphere colonization, barley germination, and poplar tree killing assays. Seven previously uncharacterized virulence genes (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150, and PA4295) were identified. The role of PA2663, a hypothetical protein discovered in the microarrays of P. aeruginosa while killing poplar trees, was examined in further detail. Expression of PA2663 protein increases biofilm formation in P. aeruginosa PAO1 drastically. By complementing the PA2663 mutation in trans and by studying with DNA microarrays and RT-PCR the PA2663 mutant vs. the wild-type strain, PA2663 was confirmed to be related to biofilm formation and was found that it is the first protein to control the psl operon in P. aeruginosa PAO1. Furthermore, PA2663 protein increases pyoverdine synthesis and quorum sensing (QS)- regulated phenotypes. A biofilm formation-related hypothetical protein, PA0939, was identified in this study. The effects of indole and 7-hydroxyindole on P. aeruginosa virulence factors were also examined for the first time. Indole and 7HI repressed expression of mexGHI-opmD multidrug efflux pump genes and genes involved in synthesis of QS-regulated virulence factors (pyocyanin, rhamnolipid, PQS, and pyoverdine production). In addition, the effects of an anti-cancer uracil analog, 5-fluorouracil (5-FU) on P. aeruginosa virulence factors and E. coli K-12 biofilm formation were examined. 5-FU repressed biofilm formation, abolished quorum-sensing phenotypes, and reduced virulence in P. aeruginosa. DNA microarray and biofilm studies with 5-FU in E. coli revealed that 5-FU controls biofilm formation through the AriR protein in E. coli K-12 strain. The effects of lsrR and lsrK mutations on E. coli biofilm formation were also examined by flow cell experiments.Item Interplay between promoter occupancy and chromatin remodeling requirements in transactivation of the S.cerevisiae PHO5 gene(Texas A&M University, 2006-04-12) Dhasarathy, ArchanaIn higher eukaryotes, DNA is packaged with histones and other proteins into chromatin. While this is important in the control of unwanted gene expression, chromatin also serves as a barrier to many vital functions in the cell. Therefore, cells have evolved many different types of chromatin remodeling enzymes to contend with this inhibitory structure and enable gene expression and other functions. The Saccharomyces cerevisiae PHO5 gene is triggered in response to phosphate starvation. In this study, I evaluate the chromatin remodeling requirements of this gene with respect to the multisubunit complexes SWI/SNF and SAGA. I show, for the first time, physical recruitment of SWI/SNF to the PHO5 promoter. I also demonstrate the role of promoter occupancy in influencing requirements for chromatin remodeling enzymes. Further, I describe various interactions between these two complexes at the PHO5 promoter. This study presents evidence for the first instance of excess recruitment of an ATP-dependent remodeler potentially compensating for the lack of a histone acetyltransferase.Item Role of two secreted proteins from Trichoderma virens in mycoparasitism and induction of plant resistance(Texas A&M University, 2007-04-25) Djonovic, SlavicaThe soil-borne filamentous fungus Trichoderma virens is a biocontrol agent with a well known ability to produce antibiotics, parasitize pathogenic fungi and induce systemic resistance in plants. Here we report the identification, purification and characterization of an elicitor secreted by T. virens; a small protein designated Sm1 (small protein 1). Confrontation and disk assays demonstrated that Sm1 lacks toxic activity against plants and microbes. Native, purified Sm1 triggers production of reactive oxygen species in rice (Oryza sativa) and cotton (Gossypium hirsutum), and induces the expression of defense related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development and is transcriptionally regulated by nutrient conditions and the presence of a host plant. When T. virens was co-cultured with cotton in an axenic hydroponic system, SM1 expression and secretion of the protein was significantly higher than when the fungus was grown alone. These results indicate that Sm1 is involved in plant-Trichoderma recognition and the induction of resistance by activation of plant defense mechanisms. Following the cloning of SM1, strains disrupted in or over-expressing SM1 were generated. Targeted gene disruption revealed that SM1 was not involved in fungal development. Expression of defense related genes in cotton and maize (Zea mays) was induced locally and systemically following colonization by T. virens in the hydroponic system. Low levels of expression of cotton or maize defense genes were found when seedlings were grown with a T. virens strain disrupted in SM1, ssupporting the Sm1-elicitor hypothesis. Additionally, unique proteins in T.virens-cotton/maize interaction were identified. Thus, the induction of defense responses in two agriculturally important crops appears to be microbially mediated. Functional analysis of a cell wall degrading enzyme, beta-1,6-glucananse (Tv-bgn3) from T. virens, demonstrated involvement of this enzyme indirectly in mycoparasitic activity of T. virens. Protein extracts from the strain disrupted in TV-BGN3 displayed reduced capability to inhibit growth of Pythium ultimum as compared to the wild-type. Additionally, protein extracts from the strains co-expressed with TV-BGN2 (beta-1,3-glucananse) from T. virens showed a significantly increased capability to inhibit growth of P. ultimum and Rhizoctonia solani hyphae.Item Role of Vaccination in the Control of Turkey Coccidiosis: Vaccine Associated Oocyst Shedding, Lesions, and Mucosal Gene Expression(2012-07-11) Behl, Michelle 1983-Coccidiosis vaccine associated side effects, oocyst shedding patterns, intestinal lesions, and mucosal gene expression in the turkey were studied. The first study examined vaccine associated side effects and oocyst shedding patterns under experimental conditions. Peak oocyst shedding occurred on days 5-6, 13-17, and 19-20 days post vaccination. Throughout the course of the study, several poults exhibited clinical coccidiosis. Based on body weights, growth was correlated with vaccine cycling. The second study examined coccidiosis vaccine induced lesions and changes in mucosal gene expression on day 5, 10, 13, 17, and 20 days post vaccination. Poults were gavaged the equivalent of 0x, 1/2x, 1x, and 2x the available vaccine dose. Intestinal sections adjacent to the Meckel's diverticulum, ileocecal junction, and middle of the ceca were collected for histological analysis and gene expression. Measurements from the tip of the villus to the base of the lamina propria, villus width, and the muscularis mucosae thickness were acquired from the histological sections. Interleukin-10, IL-1beta, and GAPDH gene expression were measured by extracting mRNA in the tissues and quantified using real-time RT-qPCR. Starting on day five post vaccination, the control group weighed significantly more than the group that received the 2x dose. Body weight and oocyst dose were inversely related through day 17. Intestinal measurements did not necessarily correlate with the vaccine dose, although there appears to be some correlation on day five. There were no significant changes in the mucosal gene expression of IL-10 and IL-1beta in the intestinal tissue adjacent to the Meckel's diverticulum throughout the course of the study. On day five post vaccination, IL-10 and IL-1beta were significantly upregulted in the ileocecal junction. Interleukin-10 was significantly upregulated on day 17 and IL-1beta was significanlty down regulated on day 20 in the ileocecal junction. Both IL-10 and IL-1beta were significantly upregulated in the ceca days 5, 10, and 13 post vaccination. Interleukin-10 was significnalty upregulated in the ceca on day 17 and significantly down regulated on day 20. Individual variation among poults in the same group merits further attention.Item Studies on gene expression profiling in JB6 cells susceptible and resistant to tumor promoter induced neoplastic transformation and regulation of gene expression at the AP-1 DNA binding site(Texas A&M University, 2005-11-01) Samuel, ShaijaGene expression underlies all important biological processes in a cell and mis-regulated gene expression plays a causal or contributory role in several diseases including cancers. Towards identifying molecular determinants that confer susceptibility and resistance to tumor promoter induced neoplastic transformation, we analyzed the gene expression profile differences among tumor promoter TPA treated and untreated mouse epidermal JB6 cells by means of cDNA microarray analyses. The expression patterns for several genes were validated by real time PCR analyses. Seventy-four genes belonging to six functional categories were found to be differentially expressed. Data from this study implicate pathways which mediate cell adhesion, migration and interferon signalling, tumor suppressors, apoptotic proteins and transcription factors and includes twenty-six genes whose involvement has not been previously implicated in cancer. In a second study we used a DNA affinity chromatography based assay to purify two proteins that bound specifically to the AP-1 DNA binding site. Analyses of the purified proteins by mass spectrometric sequencing determined the identities of these proteins as nucleolin and Y-box binding protein 1 (YB-1). We tested the hypothesis that these proteins regulate transactivation at the AP-1 site. Overexpression of nucleolin and YB-1, both alone or in combination, repressed AP-1 dependent gene transactivation. To understand the mechanism of transrepression, we analyzed whether nucleolin and/or YB-1 affected the levels and/or disrupted the intracellular localization of the AP-1 protein subunits. Western blot analyses of all the AP-1 subunits revealed that the levels of AP-1 were unaffected. Cell fractionation confirmed that the AP-1 levels were not altered in the nuclear or cytoplasmic compartments. We further tested the hypothesis that nucleolin and YB-1 repressed AP-1 transactivation by competing with AP-1 proteins for the AP-1 site. The results from this experiment were inconclusive and the precise mechanism of repression is currently under investigation.Item Testicular function in normal and poor semen quality stallions(Texas A&M University, 2006-04-12) Bryan, Tina MichelleThe chromosomal location of endocrine genes was established, and relationships between expression of specific endocrine genes and measures of testis function in normal and poor semen quality stallions was assessed. Consensus primer sequences for glucocorticoid receptor (GR) and luteinizing hormone receptor (LHR) were used to screen the CHORI-241 equine bacterial artificial chromosome (BAC) library. The identity of PCR-positive BAC clones was confirmed by sequencing. Verified BACs were mapped to horse metaphase chromosome spreads by fluorescence in situ hybridization (FISH). The BACs containing the GR and LHR were localized by FISH to ECA 14q16-q21 and ECA15q22-q23, respectively. In addition to FISH mapping, the 5000rad horse x hamster radiation hybrid (RH) panel was screened in duplicate. Two-point linkage analysis placed GR 0 cR from LEX047, while LHR was 36.67 cR from TKY011 on ECA14 and ECA15, respectively. Total testicular parenchymal weight, mean daily sperm production (DSP) per gram parenchyma and mean apoptotic rate (406.05 ?? 24.33g vs. 180.01 ?? 34.41g, 15.29 ?? 0.87 vs. 10.24 ?? 1.10, 6.70 ?? 0.88 vs. 14.25 ?? 1.11, respectively) differed (P<0.05) between normal (n=8) and poor semen quality (n=5) stallions. Also, plasma estradiol and inhibin concentrations were higher (P<0.05) in normal stallions than in poor semen quality stallions. Testicular expression of estrogen receptor beta (ER beta), βB inhibin, prolactin receptor (PRLR), growth hormone receptor (GHR) and insulin-like growth factor I receptor (IGF-IR) mRNAs were all lower (P<0.05) in poor semen quality stallions than in normal stallions. The BACs and primers developed in this study will facilitate future investigations of GR and LHR gene structure in the horse as well as providing a resource for physiological investigation of these two genes that are primary regulators of stress responsiveness and fertility. These data add important endocrine genes to the horse cytogenetic map. Also, important hormonal and gene expression changes have been identified in poor semen quality stallions for further investigation.Item The Molecular and Physiological Basis for Temperature Mediated Regulation of Dwarfness in Tifdward Bermudagrass(2014-04-03) Abernathy, ScottTifdwarf (C. dactylon (L.) Pers. x C. transvaalensis (Burtt-Davies) has been used on putting greens in the southern US for over 50 years. Dwarfism in Tifdwarf (TD) bermudagrass is a conditional trait. Tifdwarf internodes and leaves elongate when exposed to suboptimal temperatures. This study further quantified physiological aspects of this response and investigated the role of gibberellins in the temperature mediated release of TD dwarfism. In controlled environment studies, TD internode and leaf lengths were two times longer in suboptimal (27?C/19?C day/night) compared to optimal temperatures (35/27?C). In NuMex Sahara (NM), a non-dwarf bermudagrass, internode and leaf length decreased or showed no response to suboptimal temperatures. When grown under suboptimal temperatures, TD accumulated the same or less biomass than optimal treatments. NM accumulated less biomass. Suboptimal temperature reduced respiration in TD but had no affect on photosynthesis. To investigate the role of gibberellins in conditional dwarfism, expression patterns for GA20ox1, GA20ox2, GA3ox, GA2oxa, GA2oxb and GAMyb were analyzed. Under optimal temperatures, GA20ox2 and GA3ox expression were higher and GA2oxa expression was lower in TD than NM. Similar expression patterns are common in many GA associated dwarf mutants. Despite limited phenotypic differences in NM given different temperature treatments, GA20ox2 and GA3ox were elevated and GA2oxa and GAMyb were depressed in suboptimal treatments. Unlike NM, and despite robust phenotypic changes, TD displayed minimal molecular responses to suboptimal temperatures. Only GA2oxa and GA2oxb displayed differential expression patterns between treatments. Both were higher in the suboptimal temperature regime. The GA biosynthetic inhibitors CCC and flurprimidol decreased TD internode length while GA3 increased length under both temperature treatments, however internodes from suboptimal treatments remained longer than optimal treatments. Trinexapac-ethyl also decreased internode length in both temperature treatments, but at the high application rate, no difference was measured between temperature treatments. Therefore, functional late-stage GA metabolic and/or catabolic enzymes are required for temperature mediated adjustments in TD morphology. No difference due to temperature was observed in bermudagrass internode length when an inhibitor combination plus GA3 was applied. This suggests that the temperature mediated adjustments in morphology are not the result of altered GA sensitivity.Item Transcriptional analysis of chicken immune cells following exposure to 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD)(Texas A&M University, 2006-04-12) Puebla-Osorio, NahumIn the present investigation, microarray analysis was used to identify potential TCDD gene targets. Three microarray experiments were performed to study the effect of TCDD in an established chicken B-cell line (DT40), in a chicken macrophage cell line (HD11), and in the bursa of Fabricius from embryos exposed in ovo at 6 days of incubation. From the DT40 microarray analyses, clones with sequence similarity to the apoptotic genes caspase 8 and caspase 9, and the transcription factor NFΜB, among others, were identified. Real-time quantitative polymerase chain reaction (RT-PCR) revealed that TCDD elicits aryl hydrocarbon receptor (AhR)-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3 (see chapter III). During the course of the HD11 microarray analyses, a consistent down-regulation of the matrix metalloprotease MMP-2 was observed. This finding was the basis for the hypothesis that TCDD has an effect on the gene expression of the MMP-2 and MMP-9 in macrophages. Then, gene expression analysis and functional zymography showed that TCDD impairs the MMP-2 and MMP-9 response to LPS stimulation in HD11 chicken macrophages (see chapter V). The microarray analyses of the embryonic bursa of Fabricius provided the basis to further study of the effect of TCDD in the chicken embryo. The shifted genes were classified according to their function. The down-regulated genes included: precursor of matrix metalloprotease-inhibitor, histone acyl-transferase 1, homeobox protein CUX-2, Death Associated Protein Kinase, and UDPglucosyl transferase, among others. The up-regulated genes included: phosphoinositidespecific phospholipase, acyl Co-A oxidase, and protein effector of Cdc42, among others. Together, these microarray analyses produced a database of genes of interest that will provide sufficient hypotheses to inspire multiple investigations aimed at confirming and refining the gene expression alterations as a consequence of TCDD exposure.Item Transcriptional Profiling of Immune Responses in Cattle Experimentally Infested with Amblyomma americanum ticks(2013-07-25) Brannan, Jaime LynetteInfestation of cattle by Lone Star ticks, Amblyomma americanum, leads to damage of hides intended for leather, weight loss, infertility, and potentially death of cattle, which contribute to production losses for farmers. Public concerns regarding chemical residues in food and the environment necessitate development of chemical-free alternative tick controls, such as breeding for tick-resistant phenotypes and developing anti-tick vaccines. Thus, the goal of this study was elucidation of mechanisms that mediate immune responses in cattle infested with A. americanum using gene expression techniques. Methods for isolation of total RNA from bovine tick bite-site biopsies and blood leukocytes were optimized to provide RNA suitable for gene expression studies. Tick bite-site biopsies (6 mm) and blood leukocytes were collected from a total of 13 calves (N=6, Group 4 and N=7, Group 5 calves) during experimental tick infestations to determine A. americanum tick-susceptible and -resistant phenotypes. Microarray experiments compared gene expression in tick bite-sites from tick-susceptible, moderately tick-resistant, and highly tick-resistant calves. A total of 35 genes were profiled in tick bite-site biopsies and 12 genes were evaluated in blood leukocytes via gene-specific qRT-PCR assays, and analyzed for each phenotype and for each group of calves as a whole. Analysis of microarray data revealed differential expression of IL-1R-mediators among the three cattle phenotypes. Expression profiles generated by qRT-PCR for TLR-mediating genes such as TLR2, TLR4, CD14, and MyD88 suggest that a MyD88-dependent signaling pathway may mediate the development of acquired immunity in cattle infested with Lone Star ticks. Additionally, increased expression for IL12, IFNgamma, and TNFalpha suggests that a Th1-type cell-mediated reaction may be activated, whereas increased expression of IL6, IL10, and IGHG1 supports the involvement of a Th2-type humoral-mediated response at tick bite-sites in cattle infested with at A. americanum. Regression analyses identified strong correlations between factors involved in pattern recognition in tick bite-site biopsies, including associations between TLR4 and IL1alpha, and between IL1alpha and IL1RN. In conclusion, this dissertation reports optimal methodology for gene expression studies in tick-infested cattle and provides preliminary data concerning the underlying mechanisms associated with the immune response in Lone Star tick-infested cattle.