911 resultados para 060405 Gene Expression (incl. Microarray and other genome-wide approaches)


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The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

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Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

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Mechanical forces are essential for connective tissue homeostasis. The extracellular matrix (ECM) plays a key role in the transmission of forces generated by the organism (e.g. muscle contraction) and externally applied (e.g. gravity). The expression of specific ECM proteins such as collagens and tenascin-C, as well as of matrix metalloproteinases, involved in their turnover, is influenced by mechanical stimuli. The precise mechanisms by which mechanical strains are translated into chemical signals and lead to differential gene expression are however not fully understood. Cell-matrix adhesion sites are good candidates for hosting a "mechanosensory switch", as they transmit forces from the ECM to the cytoskeleton and vice versa by physically linking the cytoskeleton to the ECM. Integrins, transmembrane proteins located to these adhesion sites, have been shown to trigger a set of internal signaling cascades after mechanical stimulation. We have shown that the expression level of tenascin-C directly correlates with externally applied mechanical stress, as well as with RhoA/RhoA-dependent kinase-mediated cytoskeletal tension. Presumably other genes are regulated in a similar manner. The changes in ECM composition and mechanical properties derived from mechanical stress are relevant in medical intervention after ligament and tendon injury.

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Acute administration of a mononclonal antibody (mAb) raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after spinal cord injury (SCI) in the rat greatly improves neurological outcomes. We have profiled gene expression in anti-CD11d and isotyped-matched control mAb-treated rats after SCI. Microarray analysis demonstrated reduced expression of pro-inflammatory cytokines and increased expression of inflammatory mediators that promote wound healing and the expression of scar proteins predicted to improve nerve growth. These changes in gene expression may reflect changes in the types of macrophages that populate the lesions in anti-CD11d mAb-treated rats.

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BDE-47 is one of the most widely found congeners of PBDEs in marine environments. The potential immunomodulatory effects of BDE-47 on fish complement system were studied using the marine medaka Oryzias melastigma as a model fish. Three-month-old O. melastigma were subjected to short-term (5 days) and long-term (21 days) exposure to two concentrations of BDE-47 (low dose at 290 +/- 172 ng/day; high dose at 580 +/- 344 ng/day) via dietary uptake of BDE-47 encapsulated in Artemia nauplii. Body burdens of BDE-47 and other metabolic products were analyzed in the exposed and control fish. Only a small amount of debrominated product, BDE-28, was detected, while other metabolic products were all under detection limit. Transcriptional expression of six major complement system genes involved in complement activation: C1r/s (classical pathway), MBL-2 (lectin pathway), CFP (alternative pathway), F2 (coagulation pathway), C3 (the central component of complement system), and C9 (cell lysis) were quantified in the liver of marine medaka. Endogenous expression of all six complement system genes was found to be higher in males than in females (p < 0.05). Upon dietary exposure of marine medaka to BDE-47, expression of all six complement genes were downregulated in males at day 5 (or longer), whereas in females, MBl-2, CFP, and F2 mRNAs expression were upregulated, but C3 and C9 remained stable with exposure time and dose. A significant negative relationship was found between BDE-47 body burden and mRNA expression of C1r/s, CFP, and C3 in male fish (r = -0.8576 to -0.9447). The above findings on changes in complement gene expression patterns indicate the complement system may be compromised in male O. melastigma upon dietary exposure to BDE-47. Distinct gender difference in expression of six major complement system genes was evident in marine medaka under resting condition and dietary BDE-47 challenge. The immunomodulatory effects of BDE-47 on transcriptional expression of these complement components in marine medaka were likely induced by the parent compound instead of biotransformed products. Our results clearly demonstrate that future direction for fish immunotoxicology and risk assessment of immunosuppressive chemicals must include parallel evaluation for both genders.

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BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

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Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of insects. IBH is a multifactorial disease with contribution of genetic and environmental factors. Candidate gene association analysis of IBH was performed in a group of 89 Icelandic horses all born in Iceland and imported to Europe. Horses were classified in IBH-affected and non-affected based on clinical signs and history of recurrent dermatitis, and on the results of an in vitro sulfidoleukotriene (sLT)-release assay with Culicoides nubeculosus and Simulium vittatum extract. Different genetic markers were tested for association with IBH by the Fisher's exact test. The effect of the major histocompatibility complex (MHC) gene region was studied by genotyping five microsatellites spanning the MHC region (COR112, COR113, COR114, UM011 and UMN-JH34-2), and exon 2 polymorphisms of the class II Eqca-DRA gene. Associations with Eqca-DRA and COR113 were identified (p < 0.05). In addition, a panel of 20 single nucleotide polymorphisms (SNPs) in 17 candidate allergy-related genes was tested. During the initial screen, no marker from the panel was significantly (p < 0.05) associated with IBH. Five SNPs associated with IBH at p < 0.10 were therefore used for analysis of combined genotypes. Out of them, SNPs located in the genes coding for the CD14 receptor (CD14), interleukin 23 receptor (IL23R), thymic stromal lymphopoietin (TSLP) and transforming growth factor beta 3 (TGFB3) molecules were associated with IBH as parts of complex genotypes. These results are supported by similar associations and by expression data from different horse populations and from human studies.

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Insufficient feed intake during early lactation results in elevated body fat mobilization to meet energy demands for milk production. Hepatic energy metabolism is involved by increasing endogenous glucose production and hepatic glucose output for milk synthesis and by adaptation of postcalving fuel oxidation. Given that cows differ in their degree of fat mobilization around parturition, indicated by variable total liver fat concentration (LFC), the study investigated the influence of peripartum fat mobilization on hepatic gene expression involved in gluconeogenesis, fatty acid oxidation, ketogenesis, and cholesterol synthesis, as well as transcriptional factors referring to energy metabolism. German Holstein cows were grouped according to mean total LFC on d 1, 14, and 28 after parturition as low [<200mg of total fat/g of dry matter (DM); n=10], medium (200-300 mg of total fat/g of DM; n=10), and high (>300 mg of total fat/g of DM; n=7), indicating fat mobilization during early lactation. Cows were fed total mixed rations ad libitum and held under equal conditions. Liver biopsies were taken at d 56 and 15 before and d 1, 14, 28, and 49 after parturition to measure mRNA abundances of pyruvate carboxylase (PC); phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; propionyl-coenzyme A (CoA) carboxylase α; carnitine palmitoyl-transferase 1A (CPT1A); acyl-CoA synthetase, long chain 1 (ASCL1); acyl-CoA dehydrogenase, very long chain; 3-hydroxy-3-methylglutaryl-CoA synthase 1 and 2; sterol regulatory element-binding factor 1; and peroxisome proliferator-activated factor α. Total LFC postpartum differed greatly among cows, and the mRNA abundance of most enzymes and transcription factors changed with time during the experimental period. Abundance of PC mRNA increased at parturition to a greater extent in high- and medium-LFC groups than in the low-LFC group. Significant LFC × time interactions for ACSL1 and CPT1A during the experimental period indicated variable gene expression depending on LFC after parturition. Correlations between hepatic gene expression and performance data and plasma concentrations of metabolites and hormones showed time-specific relations during the transition period. Elevated body fat mobilization during early lactation affected gene expression involved in gluconeogenesis to a greater extent than gene expression involved in lipid metabolism, indicating the dependence of hepatic glucose metabolism on hepatic lipid status and fat mobilization during early lactation.

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In the United States, endometrial cancer is the leading cancer of the female reproductive tract. There are 40,100 new cases and 7,470 deaths from endometrial cancer estimated for 2008 (47). The average five year survival rate for endometrial cancer is 84% however, this figure is substantially lower in patients diagnosed with late stage, advanced disease and much higher for patients diagnosed in early stage disease (47). Endometrial cancer (EC) has been associated with several risk factors including obesity, diabetes, hypertension, previously documented occurrence of hereditary non-polyposis colorectal cancer (HNPCC), and heightened exposure to estrogen (25). As of yet, there has not been a dependable molecular predictor of endometrial cancer occurrence in women with these predisposing factors. The goal of our lab is to identify genes that are aberrantly expressed in EC and may serve as molecular biomarkers of EC progression. One candidate protein that we are exploring as a biomarker of EC progression is the cell survival protein survivin.

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Brain tumor is one of the most aggressive types of cancer in humans, with an estimated median survival time of 12 months and only 4% of the patients surviving more than 5 years after disease diagnosis. Until recently, brain tumor prognosis has been based only on clinical information such as tumor grade and patient age, but there are reports indicating that molecular profiling of gliomas can reveal subgroups of patients with distinct survival rates. We hypothesize that coupling molecular profiling of brain tumors with clinical information might improve predictions of patient survival time and, consequently, better guide future treatment decisions. In order to evaluate this hypothesis, the general goal of this research is to build models for survival prediction of glioma patients using DNA molecular profiles (U133 Affymetrix gene expression microarrays) along with clinical information. First, a predictive Random Forest model is built for binary outcomes (i.e. short vs. long-term survival) and a small subset of genes whose expression values can be used to predict survival time is selected. Following, a new statistical methodology is developed for predicting time-to-death outcomes using Bayesian ensemble trees. Due to a large heterogeneity observed within prognostic classes obtained by the Random Forest model, prediction can be improved by relating time-to-death with gene expression profile directly. We propose a Bayesian ensemble model for survival prediction which is appropriate for high-dimensional data such as gene expression data. Our approach is based on the ensemble "sum-of-trees" model which is flexible to incorporate additive and interaction effects between genes. We specify a fully Bayesian hierarchical approach and illustrate our methodology for the CPH, Weibull, and AFT survival models. We overcome the lack of conjugacy using a latent variable formulation to model the covariate effects which decreases computation time for model fitting. Also, our proposed models provides a model-free way to select important predictive prognostic markers based on controlling false discovery rates. We compare the performance of our methods with baseline reference survival methods and apply our methodology to an unpublished data set of brain tumor survival times and gene expression data, selecting genes potentially related to the development of the disease under study. A closing discussion compares results obtained by Random Forest and Bayesian ensemble methods under the biological/clinical perspectives and highlights the statistical advantages and disadvantages of the new methodology in the context of DNA microarray data analysis.

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Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.

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Retinoids are known to inhibit proliferation of and induce terminal differentiation of many normal and transformed cells. It has been postulated that retinoids exert their effect by altering gene expression. HL-60 cells and macrophages both respond to retinoic acid action by the rapid induction of the enzyme tissue transglutaminase. The induction has been shown to be due to increased transcription of the transglutaminase gene. The first part of the dissertation studied the structure-function relationship of retinoid-regulated transglutaminase induction, differentiation and proliferation in HL-60 cells using retinoid analogs. The results indicated strict structural constraints and a strong structure-function correlation between transglutaminase induction and differentiation; those retinoids that induced transglutaminase also induced differentiation, those analogs that did not induce transglutaminase could not induce differentiation. The ability of the retinoids to induce transglutaminase in HL-60 cells was paralleled in macrophages. However, the antiproliferative effect of the retinoids displayed less stringent structural constraints than their differentiation- and transglutaminase-inducing properties. Specifically all the retinoids were able to inhibit proliferation to varying extents. It is concluded that the induction of transglutaminase and of differentiation by retinoids is mediated by receptors. While receptor mediation cannot be entirely ruled out, with the current data no definitive statement can be made about the antiproliferative activity of retinoids. Also, the concordance in the ability of the retinoids to induce transglutaminase and the ability to induce differentiation of HL-60 cells suggests that the former is an early response of the cells to retinoids and differentiation a later consequence on the same pathway. Using the induction of transglutaminase as an index of the direct, or primary, effect of retinoids on gene expression, the second part of the dissertation investigates, by 2D gel electrophoresis, the alteration in the rates of synthesis of other proteins in macrophages and HL-60 cells in response to short incubations with retinoic acid. Any changes in parallel with transglutaminase were taken to indicate proteins directly under the control of retinoic acid. It is concluded that retinoic acid regulates the expression of a circumscribed set of genes in a cell-specific manner. The results support the hypothesis that retinoids exert their multiple effects on myeloid cells, in part, by receptor-mediated alternations in gene expression. ^

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Histone gene expression is replication-independent during oogenesis and early embryogenesis in amphibians; however, it becomes replication-dependent during later embryogenesis and remains replication-dependent through adulthood. In order to understand the mechanism for this switch in transcriptional regulation of histone gene expression during amphibian development, linker-scanning mutations were made in a Xenopus laevis H2B histone gene promoter by oligonucleotide site-directed mutagenesis and assayed by microinjection into oocytes and embryos. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CCAAT, and ATF motifs, required for maximal transcription in both oocytes and embryos. Factors binding to the CCAAT and ATF motifs are present in oocytes and embryos and increase slightly in abundance during early development. A sequence (CTTTACAT) in the frog H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is additionally required for maximal H2B transcription in frog embryos. Oocytes and embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer-binding proteins were isolated from Xenopus cDNA libraries by virtue of their similarity with the DNA binding (POU) domain of the ubiquitously expressed transcription factor Oct-1. The protein encoded by one of these genes, termed Oct-60, was localized mainly in the cytoplasm of oocytes and was also present in early embryos until the gastrula stage of development. Proteins encoded by the other two genes, Oct-25 and Oct-91, were present in embryos after the mid-blastula stage of development and decreased by early neurula stage. The activity of the Xenopus H2B octamer motif in embryos is not specifically associated with increased binding by Oct-1 or the appearance of novel octamer-binding proteins but requires the presence of an intact CCAAT motif. We found that synergistic interactions among promoter elements are important for full H2B promoter activity. The results suggest that transcription of the Xenopus H2B gene is replication-dependent when it is activated at the mid-blastula stage of development and that replication-dependent H2B transcription is mediated by Oct-1. ^

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Cell differentiation are associated with activation of cell lineage-specific genes. The $LpS{\it 1}\beta$ gene of Lytechinus pictus is activated at the late cleavage stage. $LpS{\it 1}\beta$ transcripts accumulate exclusively in aboral ectoderm lineages. Previous studies demonstrated two G-string DNA-elements, proximal and distal G-strings, which bind to an ectoderm-enriched nuclear factor. In order to define the cis-elements which control positive expression of the $LpS{\it 1}\beta$ gene, the regulatory region from $-$108 to +17 bp of the $LpS{\it 1}\beta$ gene promoter was characterized. The ectoderm G-string factor binds to a G/C-rich region larger than the G-string itself and the binding of the G-string factor requires sequences immediately downstream from the G-string. These downstream sequences are essential for full promoter activity. In addition, only 108 bp of $LpS{\it 1}\beta\ 5\sp\prime$ flanking DNA drives $LpS{\it 1}\beta$ gene expression in aboral ectoderm/mesenchyme cells. Therefore, for positive control of $LpS{\it 1}\beta$ gene expression, two regions of 5$\sp\prime$ flanking DNA are required: region I from base pairs $-$762 to $-$511, and region II, which includes the G/C-rich element, from base pairs $-$108 to $-$61. A mesenchyme cell repressor element is located within region I.^ DNA-binding proteins play key roles in determination of cell differentiation. The zinc finger domain is a DNA-binding domain present in many transcription factors. Based on homologies in zinc fingers, a zinc finger-encoding gene, SpKrox-1, was cloned from S. purpuratus. The putative SpKrox-1 protein has all structural characteristics of a transcription factor: four zinc fingers for DNA binding; acidic domain for transactivation; basic domain for nuclear targeting; and leucine zipper for dimerization. SpKrox-1 RNA transcripts showed a transient expression pattern which correlates largely with early embryonic development. The spatial expression of SpKrox-1 mRNA was distributed throughout the gastrula and larva ectodermal wall. However, SpKrox-1 was not expressed in pigment cells. The SpKrox-1 gene is thus a marker of a subset of SMCs or ectoderm cells. The structural features, and the transient temporal and restricted spatial expression patterns suggest that SpKrox-1 plays a role in a specific developmental event. ^