Project description:Cancer cells have abnormal gene expression patterns, however, the transcription factors and the architecture of the regulatory network that drive cancer specific gene expression profiles is often not known. Here we studied a model of Ras-driven invasive tumorigenesis in Drosophila larval epithelial tissues and combined in vivo genetic analyses with high-throughput sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, we discovered that tumor specific gene expression is driven by a highly interconnected network composed of few transcription factors. These are: Stat, Mef2, the AP-4 homolog Cropped, the nuclear receptor Ftz-f1, the bHLH factors Myc and Taiman, and the AP-1 transcription factors Kayak, ATF-3, Pdp1, and dCEBPG. Many of these transcription factors are ectopically expressed and/or hyperactivated in human tumors. The members of this tumor master regulatory network are predicted to directly regulate the majority of the tumor specific gene expression profile. Similar to networks of master regulators that control organ development and cellular differentiation, there is a predicted high degree of co-regulation of target genes, and these network members are required in multiple eptihelia for tumor growth and invasiveness. We further found that Yki/Sd and bZIP/AP-1 factors, the downstream transcription factors of the Hippo and JNK pathways, initiate cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identified an ectopic yet highly ordered network of master regulators that control cancer cell specific gene expression. RNA-seq gene expression profiling across Drosophila 3rd instar larval imaginal discs (eye-antenna, wing and leg) in a hh driven tumor model, perturbations and controls.
Project description:Reactive oxygen species (ROS) are key signalling molecules that regulate growth and development and coordinate responses to biotic and abiotic stresses. ROS homeostasis is controlled through a complex network of ROS production and scavenging enzymes. Recently, the first genes involved in ROS perception and signal transduction have been identified and, currently, we are facing the challenge to uncover the other players within the ROS regulatory gene network. The specificity of ensuing cellular responses depends on the type of ROS and their subcellular production sites. Various experimental systems, including catalase-deficient plants, in combination with genome-wide expression studies demonstrated that increased hydrogen peroxide (H2O2) levels significantly affect the transcriptome of plants and are capable of launching both defence responses and cell death events. We used microarrays to assess differential gene expression provoked by H2O2 from plastid or peroxisomal origin, respectively. Columbia-0 (Col-0, wild type), catalase-deficient Salk plants (10-15% of wild-type catalase activity; cat2-2; N576998; (Queval et al., 2007)) and A. thaliana plants expressing glycolate oxidase in chloroplasts (GO5 plants; (Fahnenstich et al., 2008)) were grown in soil under a 16h light/8h dark regime at photosynthetically active photon flux densities (PPFD) of 75 µmol quanta m-2 s-1 at 22°C day/18°C night temperatures and a CO2 concentration of 3,000 ppm. After three weeks of growth, plants were transferred to ambient CO2 concentration (380 ppm) and the same PPFD. Whole rosettes were harvested at 0h and 8h after transfer. Control samples were harvested at 8 h from plants continuously maintained in high CO2.
Project description:Bile acids play multiple roles in vertebrate metabolism by facilitating lipid absorption in the intestine and acting as a signaling molecule in lipid and carbohydrate metabolism. Bile acids are also the main route to excrete excess cholesterol out of the body. Alpha-methyl-Coa racemase (Amacr) is one of the enzymes needed to produce bile acids from cholesterol. The mouse model lacking Amacr can produce only minor (less than 10%) amounts of bile acids, but still they are symptomless in normal laboratory conditions. Bile acid synthesis occurs in liver. In this experiment, liver samples from Amacr-/- and wild-type mice were collected and their gene expression levels were compared. 4 biological replicates per genotype.
Project description:Bile acids play multiple roles in vertebrate metabolism by facilitating lipid absorption in the intestine and acting as a signaling molecule in lipid and carbohydrate metabolism. Bile acids are also the main route to excrete excess cholesterol out of the body. Alpha-methyl-Coa racemase (Amacr) is one of the enzymes needed to produce bile acids from cholesterol. The mouse model lacking Amacr can produce only minor (less than 10%) amounts of bile acids, but still they are symptomless in normal laboratory conditions. Cholesterol absorption occurs in the jejunum part of the intestine. In this experiment, the intestines from Amacr-/- and wild-type mice were divided into four equal segments and the endothelial layer was collected by scraping. The RNA from these samples was isolated and the gene expression levels in each segment were compared. 3 biological replicates per genotype/segment combination.
Project description:The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulation network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach. We determined the gene expression of four different strains of E. coli on three different media. The four strains corresponded to the wild-type E. coli (LJ110), a PdhR knockout mutant (LJ110deltapdhR), a strain carrying an empty plasmid (LJ110/pTM30) and a PdhR overexpression strain (LJ110/pTM30PdhRhis). These strains were cultivated on Luria-Bertani broth (LBo), standard phosphate minimal medium supplemented with acetate and standard phosphate minimal medium supplemented with pyruvate. We obtained an overall 24 microarray experiments from two replicates of each of these cultivations.
Project description:Machado2014 - Curcumin production pathway in
This model is described in the article:
A kinetic model for curcumin
production in Escherichia coli.
Machado D, Rodrigues LR, Rocha
BioSystems 2014 Nov; 125: 16-21
Curcumin is a natural compound obtained from turmeric, and
is well known for its pharmacological effects. In this work, we
design a heterologous pathway for industrial production of
curcumin in Escherichia coli. A kinetic model of the pathway is
then developed and connected to a kinetic model of the central
carbon metabolism of E. coli. This model is used for
optimization of the mutant strain through a rational design
approach, and two manipulation targets are identified for
overexpression. Dynamic simulations are then performed to
compare the curcumin production profiles of the different
mutant strains. Our results show that it is possible to obtain
a significant improvement in the curcumin production rates with
the proposed mutants. The kinetic model here developed can be
an important framework to optimize curcumin production at an
industrial scale and add value to its biomedical potential.
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Project description:Escherichia coli DH1 cultures with treated with 6% 1,4 Butanediol for 1 hour and compared with untreated cultures The data from this experiment was used to identify a candidate for further study as described in Szmidt et al 2013 Utilizing a highly responsive gene, yhjX, in E. coli based production of 1,4-Butanediol submitted to Chemical Engineering Science 4x72k E.coli gene expression microarrays were used to study the genes that are differentialy expressed in the strain DH1 grown in defined medoin and exposed to 6% 1,4 Butanediol for one hour at mid-log growth stage.