text:
- Research
- Open Access
Multi-tissue gene-expression analysis in a mouse model of thyroid hormone resistance
© Miller et al.; licensee BioMed Central Ltd. 2004
- Received: 19 February 2004
- Accepted: 01 April 2004
- Published: 29 April 2004
Resistance to thyroid hormone (RTH) is caused by mutations of the thyroid hormone receptor β (TRβ) gene. To understand the transcriptional program underlying TRβ mutant-induced phenotypic expression of RTH, cDNA microarrays were used to profile the expression of 11,500 genes in a mouse model of human RTH.
We analyzed transcript levels in cerebellum, heart and white adipose tissue from a knock-in mouse (TRβPV/PV mouse) that harbors a human mutation (referred to as PV) and faithfully reproduces human RTH. Because TRβPV/PV mice have elevated thyroid hormone (T3), to define T3-responsive genes in the context of normal TRβ, we also analyzed T3 effects in hyperthyroid wild-type gender-matched littermates. Microarray analysis revealed 163 genes responsive to T3 treatment and 187 genes differentially expressed between TRβPV/PV mice and wild-type littermates. Both the magnitude and gene make-up of the transcriptional response varied widely across tissues and conditions. We identified genes modulated in T3-dependent PV-independent, T3- and PV-dependent, and T3-independent PV-dependent pathways that illuminated the biological consequences of PV action in vivo. Most T3-responsive genes that were dysregulated in the heart and white adipose tissue of TRβPV/PV mice were repressed in T3-treated wild-type mice and upregulated in TRβPV/PV mice, suggesting the inappropriate activation of T3-suppressed genes in RTH.
Comprehensive multi-tissue gene-expression analysis uncovered complex multiple signaling pathways that mediate the molecular actions of TRβ mutants in vivo. In particular, the T3-independent mutant-dependent genomic response unveiled the contribution of a novel 'change-of-function' of TRβ mutants to the pathogenesis of RTH. Thus, the molecular actions of TRβ mutants are more complex than previously envisioned.
- Thyroid Hormone
- White Adipose Tissue
- Adenomatous Polyposis Coli
- Additional Data File
- Average Expression Ratio
Thyroid hormone (T3) regulates growth, development and differentiation. These actions are mediated by high-affinity thyroid hormone receptors (TRs) that bind T3 and localize to the nucleus, where they regulate transcription of target genes. Four T3-binding TR isoforms, β1, β2, β3 and α1, are derived from two TR genes (β and α genes) by alternative splicing of the primary transcripts [1–3]. The expression of these TR isoforms is tissue dependent and developmentally regulated [1, 2, 4]. As transcription factors, the TRs regulate gene expression by binding thyroid-hormone response elements (TREs) in the regulatory domains of target genes which can confer specificity for TR isoforms [5]. The transcriptional activity of the TRs is also modulated by a host of co-repressors and co-activators [6].
Mutations in TRβ that affect its ability to bind T3 or interact with co-repressors results in the syndrome known as resistance to thyroid hormone (RTH) [7–9]. RTH is a dominantly inherited abnormality that manifests as an impaired sensitivity of thyroid hormone-responsive tissues to circulating T3 [7, 9]. Clinical diagnosis of RTH typically recognizes elevated levels of thyroid hormone associated with nonsuppressible thyroid-stimulating hormone (TSH) [7, 9]. The clinical presentation of the disease is hypothyroidism, with symptoms such as delayed growth, cognitive dysfunction, and hypercholesterolemia, and, concurrently, signs consistent with hyperthyroidism, including tachycardia, weight loss, attention deficit-hyperactivity disorder and advanced bone age. The hypothyroid-like effects are presumably the consequence of mutant TRβ interference with, or inhibition of, normal T3 signaling pathways, whereas the signs reflective of hyperthyroidism result from the elevated T3 driving the activity of the TRα1 isoform [10].
To investigate the physiological consequences of a mutant β receptor in the germline, a mouse model expressing a TRβ mutant was created using homologous recombination and the Cre/loxP system [11]. The targeted TRβ mutation (referred to as PV) was the same as that from a patient with severe RTH whose symptoms included elevated T3 and T4, nonsuppressible TSH, goiter, tachycardia and short stature [12]. The PV mutant was shown in vitro to lack both T3-binding activity and transcriptional capacity and to strongly interfere with the transcriptional activity of normal TRs [12]. The PV mutant was found expressed in all mouse T3-target tissues examined, including cerebrum, cerebellum, pituitary, liver, brown and white adipocytes, heart, muscle, lung, spleen and kidney [11]. TRβPV mice exhibited goiter, delayed bone development, impaired weight gain, hearing defects and hypercholesterolemia, all of which are reminiscent of the clinical presentation of RTH in humans [11, 13, 14].
The availability of this mouse model provides an extraordinary opportunity to understand the molecular actions of TRβ mutants and to elucidate the affected cellular pathways in vivo. To this end, we applied high-density cDNA microarrays to identify PV-affected genes in the heart, cerebellum and white adipose tissue (WAT) of TRβPV/PV mice. Concurrently, we analyzed the expression profiles of wild-type littermates treated with T3 to uncover T3-responsive genes in the context of normal TRβ in a model of pharmacologic hyperthyroidism. Across the three tissues analyzed, we identified (by twofold change or more) 163 distinct genes responsive to T3 treatment and 187 genes differentially expressed between TRβPV/PV mice and wild-type littermates.
The expression patterns of these genes showed a diverse transcriptional response comprising genes with tissue-specific expression and genes with similar or contrasting expression in multiple tissues. Category analysis of expression patterns identified genes that were modulated by T3-dependent PV-independent, T3- and PV-dependent, and T3-independent PV-dependent mechanisms. Intra-tissue expression analysis provided a biological glimpse of the adverse effects of PV in a tissue-dependent manner. Finally, hierarchical clustering of multi-tissue gene-expression patterns revealed evidence of discrete biological pathways including immune response, lipogenesis and cell-cycle inhibition that are modulated in multiple tissues of hyperthyroid and T3-resistant TRβPV/PV mice. These findings provide a molecular framework for understanding the variability in tissue sensitivity of RTH and provide insight into signaling pathways of mutant TRβ in vivo.
Experimental design
RTH, caused by mutation of the TRβ gene, is the consequence of abnormal transcription of thyroid-hormone-responsive genes in T3 target tissues. Using a mouse cDNA microarray containing 11,500 gene probes (representing over 10,000 unique named and unnamed expressed sequence tags (ESTs)), we profiled gene-expression patterns in mouse models of hyperthyroidism and RTH to reveal T3-responsive (hyperthyroid-associated) and mutant PV-dysregulated genes. The three T3-target tissues profiled in this study were the cerebellum, heart and WAT, all of which are important T3-target tissues in which T3-responsive genes have yet to be comprehensively analyzed. The experimental comparisons in this study included: wild-type mice versus TRβPV/PV littermates (the RTH group); wild-type mice injected with T3 versus those receiving saline injection only (the iT3 group); and wild-type mice versus a second set of wild-type animals selected in the same way (the control group).
In the RTH experiments, we identified changes in gene expression resulting from the in vivo action of PV and/or elevated T3 characteristic of clinical RTH. The iT3 experiments revealed genes that are responsive to increased levels of T3 in pharmacologic hyperthyroidism. These experiments allowed us to analyze the expression profiles in RTH that are the effects due to the increased levels of circulating thyroid hormone and thus to understand which TR-subtype-dependent transcriptional mechanisms govern gene expression. The purpose of the control group was to identify genes that naturally fluctuate as the result of normal inter-animal differences [15]. Genes identified as differentially expressed in the control group were subsequently censored in a tissue-dependent manner. For each experimental comparison, replicate microarray hybridizations were performed with reciprocal labeling (dye-swapping) to minimize technical noise (see Materials and methods).
Identification and characterization of T3-responsive genes
Differentially expressed genes (outliers) were selected based on the 2.0r criterion whereby the fold change detected on each of two reciprocally labeled arrays (r designation) was greater than twofold (up or down, but in a reciprocal manner) in at least one experimental comparison. The average expression ratios were calculated as described (see Materials and methods) and are used herein as the expression ratio measurements. To evaluate the probe-level reproducibility of the microarray data, we examined the variability of the expression ratios for all genes represented by multiple probes on the array and identified as outliers in multiple experimental comparisons (see Additional data file 1). A high degree of expression-ratio reproducibility was observed, even for changes less than twofold, lending confidence to our microarray results and outlier selection criteria. Notably, the 2.0r criterion was applied to achieve a high level of stringency in detecting gene outliers. However, this approach precludes the identification of genes that are differentially expressed at levels below 2.0r detection (for example, 1.9-fold change) thus obscuring the distinction of tissue-specific outliers and excluding potentially important distinguishing gene cassettes. In the light of this, we compiled a dataset that lists all genes passing the 2.0r criterion in at least one experimental comparison and identifies all instances (in other experimental comparisons) where these genes pass a less stringent threshold of 1.2r (see Additional data files 2 and 3). Thus, a transcript that shows a minimum change of twofold in one comparison can be subsequently screened for smaller changes, or categorized as having virtually no change in expression (that is, less than 1.2-fold), in other comparisons.
To gauge the accuracy of our methodology in correctly identifying T3-regulated genes, we searched for the 2.0r outliers across the three tissues for genes or gene products previously known to be regulated by T3. Representative genes are listed in Additional data file 4 and include 10 genes whose mRNA expression have been reported previously to be regulated by T3 and five genes that are known to be affected by T3 at the protein level. Two genes categorized as effectors are also listed. These include transthyretin (Ttr), a high-affinity serum thyroid-hormone-binding protein that transports thyroid hormone to target tissues, thus facilitating thyroid hormone distribution [16], and the retinoid X receptor α (Rxra), a member of the nuclear receptor superfamily that heterodimerizes with TRs and modulates the transcriptional activity of TRs [1].
Variability in tissue-dependent transcriptional response in mice treated with T3 and in TRβPV/PVmice
Comparison of the numbers of 2.0r outliers detected in iT3 and RTH tissues
White adipose
18 (86%)
3 (14%)
28 (29%)
70 (71%)
14 (24%)
44 (76%)
11 (65%)
6 (35%)
92 (53%)
80 (47%)
Intriguingly, the distribution patterns of up- and downregulated genes showed a consistent trend in two tissues that distinguished the hyperthyroid and RTH conditions (Table 1). In both the heart and WAT, the predominant response to T3 administration (that is, iT3 group) was mostly downregulation of expression (71% and 76% of the outliers, respectively). By contrast, in the TRβPV/PV mice we observed higher proportions of transcriptionally induced genes. In the heart and WAT of RTH animals, 65% and 53% of outliers, respectively, were upregulated genes. Together, these data support the intriguing view that the hyperthyroid phenotype is largely mediated through T3-induced suppression of gene expression, whereas RTH is underscored by an inappropriate increase in transcriptional activity in T3 target tissues. These data strongly suggest that target-tissue response to T3 administration and the RTH genotype are distinctly different.
To further characterize the target-tissue transcriptional response, we analyzed each tissue for its degree of transcriptional uniqueness. We found that in the T3-treated mice, 24% (5/21), 24% (24/98), and 22% (13/58) of outliers in the cerebellum, heart and WAT, respectively, were tissue specific (that is, differentially expressed by twofold or more in only one tissue, and not more than 1.2-fold in any other tissue). In the RTH mice, 0% (0/0), 53% (9/17), and 47% (81/172) of outliers in the cerebellum, heart and WAT, respectively, were detected in only one tissue type (data not shown). These observations reveal a considerable degree of transcriptional specificity that is dependent on tissue type, consistent with the notion that the transcriptional behavior of TRs depends on the molecular composition of the cell type with respect to the availability and levels of cofactors that modulate TR transcriptional activity [14, 18].
Gene classification by transcriptional response
The pathological hallmark of RTH is elevated thyroid hormone associated with nonsuppressible TSH. Even though the phenotypic consequences of elevated thyroid hormone in RTH patients are known, it is not clear what genes mediate the phenotypes. Furthermore, RTH may manifest as hyperthyroidism in one tissue, and, simultaneously, hypothyroidism in another [9]. Dissecting this variable phenotype into the hyperthyroid and non-hyperthyroid components at the molecular level requires a comprehensive knowledge of the hyperthyroid-related genes in each tissue, and the expression patterns of these genes in the context of RTH. Therefore, we performed a tissue-by-tissue analysis cross-comparing the genomic effects of iT3 and RTH to segregate genes according to cognate response patterns.
Category B (orange bars) includes the outlier genes that showed reversed patterns of change between iT3 and RTH mice (that is, up in iT3 but down in RTH, or vice versa). Presumably, these genes, which are responsive to increased T3 levels (in iT3 mice), are expressed in the opposite direction in TRβPV/PV mice as a consequence of mutant TRβ activity. One such outlier was identified in the cerebellum, 25 in the heart, and 33 in the WAT. Interestingly, 84% (21/25) and 79% (26/33) of these genes in the heart and WAT, respectively, were downregulated in response to T3 treatment and concomitantly upregulated in RTH, further supporting the view that an important molecular aspect of RTH is the abnormal expression of otherwise T3-repressed genes. A mechanistic explanation is that these category B genes are regulated by T3 mostly (or exclusively) through the TRβ receptor.
The majority of the outliers identified in this study, however, were found exclusively in either iT3 (category C, blue bars) or RTH (category D, pink bars) mice. Genes in category C showed a greater than twofold change in iT3 mice and less than 1.2-fold change in RTH mice. Therefore, these genes were responsive to increased T3 in the iT3 group but insensitive to elevated T3 in the presence of the mutant TRβ receptor. In this category, we identified 18, 72 and 27 such genes in the cerebellum, heart and WAT, respectively. Conversely, the outliers in category D were detected in RTH but not in the iT3 group. Compared with category C, a relatively smaller number of such genes were identified in the cerebellum (0 genes) and the heart (3 genes), but a significantly larger number of outliers (116 genes) were detected in the WAT. Taken together, these data show that the expression patterns of iT3 and RTH responsive genes are not only tissue-dependent, but also can differ markedly between the hyperthyroid and RTH states within the same tissue.
Hierarchical clustering identifies biological pathways associated with hyperthyroid (iT3) and RTH phenotypes
In the lipogenesis cluster (B, blue bar, Figure 2), six of eight genes have clearly defined roles in fatty-acid and lipid metabolism and include Fasn, Acly, Gpd1, Elovl6, Slc25a1, and a gene named 'similar to acetyl-coenzyme A carboxylase, clone IMAGE: 5151139' which has 99% identity at the protein level with rat acetyl-coenzyme A carboxylase. These genes are consistently repressed by T3 in the heart and WAT, and expressed at reduced levels in the WAT of TRβPV/PV mice, with half of the genes being induced in the heart of TRβPV/PV mice (blue bar, Figure 3). These patterns are consistent with the negative regulation of lipogenesis in the hyperthyroid heart and WAT. In RTH mice this suggests suppression of lipogenesis in the WAT with simultaneous increase in lipogenesis in the heart.
The smaller cell-cycle/growth-inhibitory cluster is composed of several genes known to inhibit growth or cell-cycle progression or promote apoptosis (C, pink bars, Figure 3). These genes include Bnip3, Gadd45a and Cdkn1a (p21). Shown adjacent to this gene cluster is the similarly expressed gene, APC (adenomatous polyposis coli), whose expression is also associated with growth inhibition (that is, via suppression of the Wnt signaling pathway which we recently showed to be negatively regulated by T3 [19]). A hallmark of these genes (with the exception of Gadd45a) is increased expression in all three tissues of the hyperthyroid mouse. Notably, p21 is also upregulated in the heart and WAT of TRβPV/PV mice, suggesting its induction in these tissues in response to high circulating T3. These findings raise the possibility that induction of growth-inhibitory mechanisms in multiple T3 target tissues may contribute to the pathogenesis of RTH.
Gene-expression patterns in hyperthyroid and RTH hearts are distinct
The heart is highly sensitive to thyroid hormone and the primary mode of T3 action is a direct influence on cardiac gene expression [20]. Increased heart rate (tachycardia) and myocardial contractility are clinical features common to both hyperthyroidism and RTH [21, 22]. As the predominant TR isoform in the heart is TRα1, and RTH patients have normal expression levels of TRα1 together with elevated thyroid hormone, it has been postulated that the cardiac effects common to both hyperthyroidism and RTH are mediated by TRα1 signaling [20, 23]. To examine this hypothesis at the molecular level, we compared the genomic effects of induced hyperthyroidism and RTH in the mouse heart. In total, we identified 105 gene outliers that showed twofold change or more in the iT3 or RTH groups (see Additional data file 5). Notably, this list does not include several genes previously implicated in T3-mediated cardiac effects such as those for the α- and β-myosin heavy chains, SERCA2 and HCN2, as they were not represented on our microarray. To focus on the set of most relevant genes in our list, we used the GO database of biological and functional gene classifications [24] to define genes known or expected to have a role in cardiac muscle contraction.
Gene-expression patterns in iT3 and RTH hearts are distinct
Clone ID and UniGene name
Muscle related
Calcium ion binding
ATP binding and electron transport
Mitochondrion related
481408 cytochrome c oxidase, subunit VIIIa
439199 aminolevulinic acid synthase 2, erythroid
571367 BCL2/adenovirus E1B 19kDa-interacting protein 1, NIP3
350881 RIKEN cDNA 5730438N18 gene
482847 uncoupling protein 3, mitochondrial
318951 malonyl-CoA decarboxylase
920211 solute carrier family 40 (iron-regulated transporter), member 1
571633 solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1
318134 translocator of inner mitochondrial membrane a
481469 [Mrps18b]
423605 aldehyde dehydrogenase 1 family, member B1
352146 ATP citrate lyase
570675 glycerol-3-phosphate dehydrogenase 1 (soluble)
554335 Rous sarcoma oncogene
1196244 lymphocyte protein tyrosine kinase
349954 myosin heavy chain 11, smooth muscle
318692 sarcolipin
604414 (vimentin)
890932 calcium channel, voltage-dependent, alpha2/delta subunit 1
479382 myosin light chain, regulatory A
354796 fibulin 1
472672 S100 calcium binding protein A10 (calpactin)
335868 (angiotensinogen)
We hypothesized that the expression patterns of these genes would be similar in the iT3 and RTH hearts as they are presumably regulated by TRα1 and responsive in the context of elevated T3. Surprisingly, however, this analysis indicated that the iT3 and RTH hearts are molecularly distinct. Of the 23 genes identified, none showed the same or similar response patterns. Eighteen genes, which ranged from fourfold induction to 20-fold suppression in the iT3 heart, showed less than a 1.2-fold change in RTH (that is, category C genes, Figure 2). Moreover, the remaining five genes were expressed in opposite directions (that is, category B genes, Figure 2). These include the gene for myosin light chain, regulatory A (> 20-fold repressed in iT3 and > 6.6-fold increased in RTH) and the sarcolipin gene (> 4.8-fold repressed in iT3 and > 3.6-fold increased in RTH).
This expression pattern trend was also apparent in the remaining 82 non-muscle-contraction-related outliers (see Additional data file 5). Of these genes, 54 showed from 2- to 11-fold change in response to T3 injection and concurrently no change in RTH mice, while only three showed a greater then twofold change in RTH with no concurrent change in iT3 mice. Of the 25 genes in this group that showed change in both iT3 and RTH mice, only five showed change in the same direction (that is, candidates for TRα1-dependent transcription, category A genes, Figure 2), whereas the remaining 20 genes showed opposing expression patterns. Such marked differences between iT3 and RTH hearts suggest a greater role for TRβ in the heart than previously envisioned, or interference of TRα1-mediated transcription by TRβPV or its downstream effects.
Functional interpretation of expression profiles in the WAT of RTH mice
Genes involved in cell adhesion, lipogenesis and immune cell biology are modulated in white adipose tissue of RTH and iT3 mice
Clone ID and UniGene name
Cell Communication: cell adhesion
484261 : keratin complex 1, acidic, gene 13
335736 : keratin complex 2, basic, gene 6a
464060 : keratin complex 1, acidic, gene 19
420709 : RIKEN cDNA 5730453H04 gene
762299 : CEA-related cell adhesion molecule 1
776133 : cadherin 1
719965 : desmocollin 2
761578 : desmoglein 2
Metabolism: lipogenesis
570675 : glycerol-3-phosphate dehydrogenase 1 (soluble)
352146 : ATP citrate lyase
439735 : Mus musculus, Similar to acetyl-coenzyme A carboxylase, clone 5151139
576881 : fatty acid synthase
693560 : ELOVL family member 6, elongation of long chain fatty acids (yeast)
578436 : phosphogluconate dehydrogenase
949810 : hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase)
Response to external stimuli: immune cell biology
620268 : CD53 antigen
621878 : selectin, lymphocyte
618271 : histocompatibility 2, class II antigen A, beta 1
747378 : histocompatibility 2, class II antigen A, alpha
597433 : chemokine (C-X-C motif) ligand 13
637849 : histocompatibility 2, Q region locus 7
748587 : histocompatibility 2, class II antigen E beta
575397 : chemokine (C-C motif) ligand 6
851752 : chemokine (C-C motif) ligand 22
832043 : chemokine (C-C motif) ligand 19
596470 : CD79B antigen
Within the metabolism class, we identified a number of genes negatively regulated in the WAT of iT3 and RTH mice that are directly involved in fatty-acid and lipid metabolism (Table 3). These genes include those previously discovered in the lipogenesis cluster of Figure 3 (that is, Fasn, Acly, Gpd1, Elovl6, Slc25a1 and IMAGE: 5151139). Fatty-acid biosynthesis is known to use large amounts of NADPH. As a consequence, adipose tissue is known to express high levels of the enzymes of the pentose phosphate pathway that generate NADPH. Hexose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are two of the three major enzymes in the pentose phosphate pathway, and both were negatively regulated in the WAT of RTH animals.
The genes assigned to the class response to external stimuli were, with only one exception, all downregulated in RTH animals, and in some cases, in iT3 mice (Table 3). All the genes identified here are involved in immune response or immune-cell biology and overlap with the previously identified immunity-gene cluster of Figure 3. They include a number of chemokines, HLA antigens and other markers of lymphoid lineages.
Taken together, these data suggest that the WAT of RTH mice is characterized by increased cell-cell adhesion via desmosomal structures, and they recapitulate our initial findings of a possible hyperthyroid-associated inhibition of lipogenesis and a repression of immunomodulatory signaling.
Gene-expression patterns in the cerebellum of iT3 and RTH mice
Thyroid hormones are essential for normal brain development and regulate neuronal proliferation, migration and synaptogenesis in the cerebellum [29, 30]. In our study, the cerebellum was the least responsive tissue in both iT3 and RTH mice, with only 13 genes and no genes, respectively showing twofold or greater changes. The complete list of genes with all GO classifications is shown in Additional data file 7. The most highly induced gene (> threefold) in the cerebellum of T3-treated mice was the T3/T4-binding protein, transthyretin, which is synthesized in large amounts in the choroid plexus and is essential for the transport of thyroid hormones from the blood to the brain [31]. Increased expression of transthyretin may represent a positive feedback mechanism for enhancing the effect of thyroid hormone in the brain. mRNA for the retinoid X receptor (RXR) was also induced more than threefold in response to T3. RXR is a member of the nuclear receptor superfamily that heterodimerizes with TRs and modulates the transcriptional activity of TRs [1]. T3-induced upregulation of RXR may reflect the relative importance of TR-RXR heterodimerization for T3 action in the cerebellum. Also induced by T3 treatment were the genes APC and dickkopf homolog 3, which are known or suspected antagonists of the Wnt signaling pathway. Both T3 and Wnt pathways are known to have important roles in neuronal growth and synaptogenesis [32–35]. We recently presented genetic and biochemical evidence that thyroid hormone can inhibit the Wnt signaling pathway [19]. Thus, T3-mediated inhibition of Wnt signaling may be an important aspect of cerebellar development and function. The physiologic impact of the activation of these genes in hyperthyroidism warrants further investigation.
In this study, we sought to define the molecular basis of the target-tissue phenotype for hereditary TRβ mutations using a global gene expression approach. Specifically, we used expression profiling as a complex readout for the in vivo actions of T3 and PV in target tissues. The animal model was a murine line with the TRβ disrupted in the identical manner as found in the human condition (TRβPV mice). This TRβPV mouse faithfully reproduces human RTH [11, 13, 14]. To study the in vivo effects of T3 in the presence of normal TRβ, we used a mouse model of pharmacologic hyperthyroidism by treating the wild-type siblings with T3 (iT3 mice). This model aided in the delineation of genes associated with the hyperthyroid and RTH phenotype. Gene-expression patterns were analyzed in three T3 target tissues - cerebellum, heart and WAT - previously shown to have marked variation in metabolic patterns after T3 treatment.
Several interesting observations emerged from our study. First, we found that the normal physiologic effect of T3 on specific tissues is predominantly downregulation of gene expression. In the T3-treated mice, the majority of expression outliers in the heart (71%) and WAT (76%) were repressed. The same phenomenon has been observed in other mouse in vivo expression studies, albeit using less comprehensive array technologies. Feng et al. [36] identified 55 outliers in the livers of hypothyroid mice injected with T3; of these, 14 (25%) were upregulated and 41 (75%) were downregulated. Similarly, in a mouse model of T3-induced involution of a thyrotrope tumor, Wood et al. [37] identified 47 T3-responsive genes: seven (15%) were upregulated whereas 40 (85%) were downregulated. Thus, taken together, these studies indicate that T3 predominantly suppresses gene expression in responsive tissues. As would be predicted, in the RTH model, the majority of genes in the heart (63%) and, to a lesser extent in the WAT (53%), were upregulated. Of the 25 genes in the heart and 33 genes in WAT that showed opposite expression patterns in RTH (PV) and iT3 mice, 84% and 79% in heart and WAT, respectively, were repressed by T3 treatment and induced in RTH. Together, these data suggest that certain RTH (and perhaps hypothyroid) tissue phenotypes may be due predominantly to the inappropriate induction of genes normally repressed by T3. Using the number of differentially expressed genes as a semi-quantitative measure of pharmacodynamic effect, we found that the cerebellum is by far the least responsive in both iT3 and RTH, whereas the heart is the most responsive in iT3 (approximately fivefold more than cerebellum and approximately twofold more than WAT; Table 1) and the WAT is the most responsive in RTH (around 10-fold more than heart, while the cerebellum showed no changes in gene expression greater than twofold; Table 1).
Category analysis of expression patterns in iT3 and RTH mice shed light on the transcriptional consequences of the PV mutant (Figure 2). Category A consists of genes for which the T3-induced response was not affected by the expression of PV, indicating that the responses of these genes to increased T3 are likely to be exclusively mediated by TRα1. However, we have recently demonstrated that PV can form inactive heterodimers with TRα1, thereby suppressing the transcriptional activity of TRα1 [10]. This suggests the possibility that genes in category A may, in some instances, be modulated by TR-independent pathways: a notion supported by recent studies demonstrating that T3 can act in alternate pathways independent of TRs [38]. Categories B and C consist of T3-response genes mediated by TRs, but their transcriptional responses are altered by PV. In category B, the directionality of response in iT3 and RTH is reversed. These genes may be of particular importance in the phenotypic manifestations of RTH. Notable examples are the TSHβ and the α-glycoprotein common subunit genes that are repressed in the mouse pituitary following T3 administration [39]. However, in the pituitary of TRβPV/PV mice, the expression of the PV mutant leads to the induction of these two genes, resulting in the highly elevated serum TSH [11].
Genes in category C were responsive to T3 treatment yet insensitive to hyperthyroid levels of T3 in the presence of PV. These genes are not differentially expressed between untreated euthyroid wild-type mice and TRβPV/PV littermates and thus may reflect changes specific to pharmacologic hyperthyroidism. Alternatively, these genes may be primary targets of TRα1 and their expression levels in TRβPV/PV mice mimic euthyroid levels as a result of partial transcriptional interference by PV. Some of the interesting genes in this category include cytochrome c oxidase, subunit VIII and Rous sarcoma oncogene (Table 2).
One particularly interesting finding concerns the genes in category D. These genes did not respond to T3 treatment, but showed altered expression in the presence of PV. WAT had a particularly high number of genes in this category (53% (116/215) compared to heart (3%) (3/105)). The modulation of these genes in RTH could arise from the systemic effects of T3 resistance. Alternatively, the transcriptional response of these genes could be mediated by PV, either directly or indirectly, in a T3-independent manner. For example, we have previously shown that PV can heterodimerize with the 9-cis retinoacid receptor, RXR [10]. Heterodimerization of a mutant TRα with other nuclear receptors could simultaneously alter other hormone signaling pathways in RTH in a T3-independent manner.
The experimental design of this study afforded a unique opportunity to investigate the involvement of cellular pathways that may contribute to the pathophysiology of mutant TRβ action in the intact animal. Through pattern analysis of genes identified as differentially expressed in iT3 or TRβPV/PV mice in one or more tissues, we identified several gene clusters with clear functional associations that may have pathological implications in thyroid hormone disease (Figure 1). Then, on a tissue-by-tissue basis, we were able to focus on expression patterns and pathways most relevant to the tissue type.
The lipogenesis cluster in Figure 3 consists of six genes (Fasn, Acly, Gpd1, Elovl6, Slc25a1 and the homolog of rat Acac) that were repressed in both iT3 and RTH mice (category A genes; Table 1). These genes are known to have important roles in fatty-acid and lipid synthesis. This finding presumably reflects the known function of T3 as a regulator of lipogenesis and lipolysis in adipocytes [26] and, specifically, as a transcriptional modulator of Fasn, Acly and Gpd1 [40–42]. Whereas T3 is known to induce lipogenesis in the liver [26], downregulation of fatty-acid synthase and acetyl-CoA carboxylase genes was reported in brown adipose tissue of T3-treated hypothyroid rats [24]. This is consistent with the physiologic findings that hyperthyroid rats display diminished fatty-acid synthesis in brown and white adipose tissues [26]. That all six of these genes were downregulated in the WAT of both iT3 and RTH mice suggests that the T3-mediated inhibition of lipogenesis in this tissue is independent of TRβ and may be the main targets of TRα1 in this tissue. This possibility is supported by the observations of multiple TR transcriptional response elements in the promoters of both Fas and Gpd1 [43, 44] and the observation that mice heterozygous for a TRα1 point mutation (resulting in a dominant-negative TRα1 mutant with elevated T3 and T4) showed a more than fourfold increase in body fat compared to wild-type mice [45]. The suppression of these genes in both T3-treated and TRβPV/PV mice could represent an important mechanism in the phenotype of body-fat reduction common to both hyperthyroidism and RTH.
We identified in Figure 3 a small cluster of genes with similar expression patterns that are composed of cell-cycle inhibitory and apoptosis-promoting genes. Cdkn1a (encoding p21, a major cyclin-dependent kinase inhibitor that induces G1 arrest), APC (a negative regulator of Wnt-induced cell proliferation), and Bnip3 (a potent promoter of apoptosis) are all induced by T3 treatment in the cerebellum, heart and WAT. The gene for the growth inhibitor Gadd45a is induced in the heart of iT3 mice and the WAT of RTH mice, in a fashion similar to that of Cdkn1a. Though the mitogenic activity of thyroid hormone has been documented in vitro [46, 47], little is known of the cellular mechanisms underlying the antiproliferative [48, 49] and apoptotic effects [50, 51] of T3. Recently, we and others have shown that T3 treatment of cultured cells can induce expression of the p53 tumor suppressor gene [19, 52]. Several of the genes in this T3-inducible cluster are known transcriptional targets of p53, including Gadd45a [53], Cdkn1a [54] and APC [55], suggesting that a coordinated transcriptional cassette supportive of growth arrest induced by T3 is likely to be mediated directly and/or indirectly by TRα1. The induction of an antiproliferative/tumor suppressor pathway is also consistent with our previous findings that the Wnt pathway is coordinately repressed by T3 in rat pituitary cells [19]. v-erbA is a viral oncogene whose oncogenic function is the inhibition of the thyroid hormone receptor through a dominant-negative effect. These observations support our hypothesis that a major mechanism for the transforming function of v-erbA is its cellular inhibition of T3/TRα1 effects, resulting in coordinate upregulation of oncogenic pathways (that is, the Wnt signaling pathway), and repression of antiproliferation and tumor suppressor signals (for example, p53, Gadd45a, Cdkn1a).
The heart is a major target of thyroid hormone action, and T3 has a direct effect on heart rate and contractile function. Both TRα1 and TRβ are expressed in cardiac myocytes; however, TRα1 is the predominant subtype, and recent reports have suggested a primary role for TRα1 in mediating T3 effects in the heart [10, 59, 60]. It has been hypothesized that, in cardiac tissue from TRβPV/PV mice, the primary effects of the elevated T3 would be mediated through heightened T3/TRα1 signaling and that the expression response would be similar, if not identical, to that found in the myocardium of wild-type mice with induced hyperthyroidism by exogenous administration of T3. Contrary to this hypothesis, we observed a large degree of discordant cardiac gene expression when we compared the responsive genes of T3-treated animals having wild-type TRα1 and TRβ with those of TRβPV/PV mice with wild-type TRα1 (Table 2). The hearts of T3-treated animals showed expression of genes indicative of an altered workload. When heart workload increases or decreases, there is a switch in energy source from predominantly the oxidation of fatty acids to the oxidation of glucose [61]. Three key regulators facilitate this switch: malonyl-CoA decarboxylase (MCD), uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4). In our study, both MCD (which regulates β-oxidation of long-chain fatty acids) and UCP3 (which uncouples the oxidative phosphorylation of ADP) were upregulated at the mRNA level in the hearts of T3-treated animals, with concommitant downregulation of all the genes of the lipogenesis cluster (Figure 3). In contrast, the RTH heart did not show induction of MCD or UCP3, but rather shows increased mRNA levels for the lipogenic genes Fasn, Elovl6 and Slc25a1, suggesting a different metabolic signaling in the RTH heart. Recently, Swanson et al. reported that the TRβPV/PV mice, under euthyroid conditions, have decreased heart rate and contractile function, demonstrating that despite a lower expression of TRβ as compared with TRα1 in the mouse heart, the homozygous PV mutant can negatively interfere with TRα1 signaling in this organ [62]. Our genomic results are in agreement with these physiologic findings and provide a glimpse at the gene-by-gene tissue response to T3 stress.
The clinical manifestations of RTH and hyperthyroidism are well documented, and although the genetic cause of RTH is known, the target-tissue transcriptional responses and cellular pathways affected are far from being understood. In our study we have used expression profiling to dissect the molecular consequences of a dominant-negative TRβ homozygous mutation in vivo, and in the process, develop a better understanding of TRα1 effects in the whole animal. Our results show that T3 primarily acts to repress gene expression, and that TRβ has a greater modulating effect in the heart than originally thought. Moreover, we identified novel physiologic candidates for more subtle T3 action such as changes in immune-gene expression, and in the induction of antiproliferative genes. Lastly, our analysis further confirms that the relative levels of TR isoforms lead to dramatic differential effects on gene expression. The results permit the identification of genetic elements that contribute to downstream isoform effects in individual tissues [59, 60].
Materials and methods
Mouse strains and treatment
The animal protocols used in the present study have been approved by the National Cancer Institute Animal Care and Use Committee. The TRβPV mice contain a cytosine insertion in exon 10 of the TRβ1 gene at nucleotide position 1,642 of the TRβ1 cDNA. This mutation leads to a frameshift of the carboxy-terminal 14 amino acids of TRβ1 [11, 12]. TRβPV mice were prepared via homologous recombination and maintained on a mixed background of 129/Sv × C57BL/6J [11]. TRβ+/+ and TRβPV/PV male siblings at 8-10 weeks of age were used in this study. Another group of wild-type (TRβ+/+) male littermates of TRβPV/PV received daily intraperitoneal injections of T3 (5 μg/mouse) or the same volume of vehicle (phosphate-buffered saline) for 7 days. On the eighth day the animals were sacrificed by CO2 exposure and tissues (cerebellum, heart and white adipose) were harvested.
RNA preparation
Total RNAs were extracted from liquid nitrogen-frozen tissue specimens using Trizol reagent according to the manufacturer's instructions (Gibco-BRL). To minimize differences in gene expression owing to inter-animal variability, for each tissue and animal category, 5 μg of total RNA from four animals were pooled before RNA amplification. RNA amplification and quality assessment was carried out as previously described [64].
Microarray design and manufacture
The mouse cDNA microarrays contained 11,520 sequences derived from various sequence-verified clone collections including the Incyte GEM1 mouse clone set (8,700; Incyte), the Research Genetics mouse sequence-verified set (2,600; Research Genetics,), and a private collection of 200 mouse ESTs (a gift from Lothar Heninghausen, NCI, Bethesda, MD). All arrays were manufactured at the NCI Microarray Facility at the Advanced Technology Center, Gaithersburg, MD. Arrays were printed using an OmniGrid microarrayer (GeneMachines) on poly-L-lysine-coated-glass according to Eisen and Brown [63].
Microarray hybridization and scanning
For each experimental comparison, duplicate microarray hybridizations were performed according to a reciprocal strategy termed dye-swapping. In this approach, the Cy3 and Cy5 labeling scheme for the two arrays are reversed such that a true differentially expressed gene having a ratio of 2.0 (that is, a 2-fold red (Cy5-biased) spot) on one array, for example, would have a reciprocal ratio of approximately 0.5 (that is, a 2-fold green (Cy3-biased) spot) on the other array. Notably, this form of experimental replication is more rigorous than conventional duplication (that is, true replicates using the same labeling scheme) as it takes into account a recognized form of experimental noise in microarray technology characterized by the reproducible bias of one fluorophore over the other at low signal intensity features (termed dye bias). Three micrograms amplified RNA were used to generate Cy3- or Cy5-labeled cDNA for each array hybridization. cDNA labeling reaction and array hybridization were carried out essentially as described [63]. Microarrays were scanned on a GenePix 4000A microarray scanner (Axon Instruments) to generate 16-bit TIFF images of Cy3 and Cy5 signal intensities. The images were analyzed using GenePix Pro 3.0 microarray analysis software to measure fluorescence signals and format data for database deposition.
Outlier selection and data analysis
All array data were deposited in the NCI-CIT microarray database [65] where Cy3 and Cy5 signals were median normalized and expression ratios (Cy5/Cy3) were calculated. The following selection criteria were applied. All spots having a mean signal (after background subtraction) less than twice that of background in both Cy3 and Cy5 channels were systematically excluded from the dataset to eliminate measurements based on poorly detected features. The data were also filtered to exclude spots flagged as missing or corrupt in one or both arrays of a dye-swap pair. Remaining outliers were then selected by the 2.0r criterion, whereby an expression ratio must demonstrate a twofold or greater change on each array of a pair of reciprocal dye-swap hybridizations. We also demanded that the ratios must be in opposite expression directions (that is, > 2.0 on one array, and < 0.5 on the other) to exclude 'false-positive outliers' that showed a reproducible bias towards either red or green fluorescence and thus failed to reciprocate in the dye-swap array pairs. We next calculated the average expression ratio from the dye-swap pairs (after taking the reciprocal ratio from one of the two arrays) and report the average expression ratios in all analyses herein. For reporting genes by name, IMAGE clone IDs corresponding to the microarray probe sequences were used to extract UniGene Cluster IDs and names (UniGene Build 129). For genes represented by multiple probes on the array, a single probe was used (that is, the probe that showed the greatest transcriptional variation) to report the expression ratio so as to prevent gene number biasing owing to redundant probes.
RT-PCR analysis
The expression of selected genes was also analyzed by quantitative RT-PCR using the following pairs of primers:
APC forward primer 5'-GCTGACATCTGTGCTGTGGATGG-3';
APC reverse primer 5'-TCCTTAAAGCTGCTGCACTTCCC-3';
somatostatin forward primer 5'-TGCATCGTCCTGGCTTTGG-3';
somatostatin reverse primer 5'-GAGTTAAGGAAGAGATATGGG-3';
tenascin C forward primer 5'-TTCGTGTGTTCGCCATCTTG-3';
tenascin C reverse primer 5'-GTGTGAGGTCGATGGTGGT-3';
carbonic anhydrase 4 forward primer 5'-TCACTGCTAGGACAAAGGTG-3';
carbonic anhydrase 4 reverse primer 5'-AGAGGTTGAATGGGGTTTGG-3';
lysyl oxidase forward primer 5'-CTACATCCAGGCTTCCACG-3';
lysyl oxidase reverse primer 5'-TCTCCTCTGTGTGTTGGCAT-3';
enolase 3β, muscle forward primer 5'-TCAAGGGTTACACTCTGCCT-3';
enolase 3β, muscle reverse primer 5'-TCGTTCACCCACACAAGGAA-3';
creatine kinase, muscle forward primer 5'-TTCCTTGTGTGGGTGAACGA-3';
creatine kinase, muscle reverse primer 5'-TTTTCCAGCTTCTTCTCCATC-3';
GAPDH forward primer 5'-ACATCATCCCTGCATCCACT-3';
GAPDH reverse primer 5'-GTCCTCAGTGAGCCCAAG-3'.
Total RNA (5 μg) prepared as described above was used in the quantitative RT-PCR reaction. cDNA was prepared by SuperScript II reverse transcriptase I (Invitrogen) in the presence of poly(dT) primer. Preliminary experiments were carried out to define the conditions under which the amplification by PCR was in the linear range. The final conditions used for each were: 94°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec followed by 72°C for 2 min. A total of 26 cycles were used for the amplification of APC, carbonic anhydrase 4, lysyl oxidase and somatostatin cDNA. A total of 32 cycles were used for the amplification of tenascin C, enolase 3β (muscle) and creatine kinase (muscle) cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis and detected by ethidium bromide staining. The intensities of the bands were quantified by Eagle Eye (Stratagene). The expression of GAPDH was determined similarly (22 cycles) as an internal control. The band intensities of the quest genes were normalized against the control. The data are expressed as mean value ± SEM (n = 3 independent determinations). Differences between groups were examined for statistical significance using Student's t-test. p < 0.05 is considered statistically significant.
Additional data files
The following additional data files are available with the online version of this article and also at the authors' website [66]: an Excel table (Additional data file 1) of all redundant microarray probes and their average expression ratio measurements; an Excel file (Additional data file 2) containing a comprehensive filtered gene list of average expression ratios; a figure (Additional data file 3) showing a hierarchical clustergram of 2.0r outlier genes with full annotation; a Word table (Additional data file 4) listing the responsive genes known to be modulated by T3; an Excel table (Additional data file 5) listing genes differentially expressed in the heart of iT3 and/or RTH mice, while another table (Additional data file 6) lists genes differentially expressed in the white adipose tissue (WAT) of iT3 and/or RTH mice. Also provided is an Excel file (Additional data file 7) listing genes that are differentially expressed in the cerebellum of iT3 and/or RTH mice; and, finally, a zipped text file (Additional data file 8) containing the raw data.
Authors' Affiliations
- Cheng SY: Multiple mechanisms for regulation of the transcriptional activity of thyroid hormone receptors. Rev Endocr Metab Disord. 2000, 1: 9-18. 10.1023/A:1010052101214.View ArticlePubMedGoogle Scholar
- Yen PM: Physiological and molecular basis of thyroid hormone action. Physiol Rev. 2001, 81: 1097-1142.PubMedGoogle Scholar
- Harvey CB, Williams GR: Mechanism of thyroid hormone action. Thyroid. 2002, 12: 441-446. 10.1089/105072502760143791.View ArticlePubMedGoogle Scholar
- O'Shea PJ, Williams GR: Insight into the physiological actions of thyroid hormone receptors from genetically modified mice. J Endocrinol. 2002, 175: 553-570.View ArticlePubMedGoogle Scholar
- Forrest D, Vennstrom B: Functions of thyroid hormone receptors in mice. Thyroid. 2000, 10: 41-52.View ArticlePubMedGoogle Scholar
- McKenna NJ, Lanz RB, O'Malley BW: Nuclear receptor coregulators: cellular and molecular biology. Endocrinol Rev. 1999, 20: 321-344. 10.1210/er.20.3.321.Google Scholar
- Refetoff S, Weiss RE, Usala SJ: The syndromes of resistance to thyroid hormone. Endocr Rev. 1993, 14: 348-399. 10.1210/er.14.3.348.PubMedGoogle Scholar
- Yoh SM, Chatterjee VK, Privalsky ML: Thyroid hormone resistance syndrome manifests as an aberrant interaction between mutant T3 receptors and transcriptional corepressors. Mol Endocrinol. 1997, 11: 470-480. 10.1210/me.11.4.470.PubMed CentralView ArticlePubMedGoogle Scholar
- Weiss RE, Refetoff S: Resistance to thyroid hormone. Rev Endocr Metab Disord. 2000, 1: 97-108. 10.1023/A:1010072605757.View ArticlePubMedGoogle Scholar
- Zhang XY, Kaneshige M, Kamiya Y, Kaneshige K, McPhie P, Cheng SY: Differential expression of thyroid hormone receptor isoforms dictates the dominant negative activity of mutant beta receptor. Mol Endocr. 2002, 16: 2077-2092. 10.1210/me.2002-0080.View ArticleGoogle Scholar
- Kaneshige M, Kaneshige K, Zhu X, Dace A, Garrett L, Carter TA, Kazlauskaite R, Pankratz DG, Wynshaw-Boris A, Refetoff S, et al: Mice with a targeted mutation in the thyroid hormone beta receptor gene exhibit impaired growth and resistance to thyroid hormone. Proc Natl Acad Sci USA. 2000, 97: 13209-13214. 10.1073/pnas.230285997.PubMed CentralView ArticlePubMedGoogle Scholar
- Parrilla R, Mixson AJ, McPherson JA, McClaskey JH, Weintraub BD: Characterization of seven novel mutations of the c-erbA beta gene in unrelated kindreds with generalized thyroid hormone resistance. Evidence for two "hot spot" regions of the ligand binding domain. J Clin Invest. 1991, 88: 2123-2130.PubMed CentralView ArticlePubMedGoogle Scholar
- Griffith A, Szymko Y, Kaneshige M, Quinonez R, Kaneshige K, Heintz K, Masteroianni M, Kelly M, Cheng SY: Knock-in mouse model for resistance to thyroid hormone (RTH): an RTH mutation in the thyroid hormone receptor β gene disrupts cochlear morphogenesis. J Assoc Res Otolaryngol. 2002, 3: 279-288. 10.1007/s101620010092.PubMed CentralView ArticlePubMedGoogle Scholar
- Kamiya Y, Zhang XY, Ying H, Kato Y, Willingham MC, Xu J, O'Malley BW, Cheng SY: Modulation by steroid receptor coactivator-1 of target-tissue responsiveness in resistance to thyroid hormone. Endocrinology. 2003, 144: 4144-4153. 10.1210/en.2003-0239.View ArticlePubMedGoogle Scholar
- Pritchard CC, Hsu L, Delrow J, Nelson PS: Project normal: defining normal variance in mouse gene expression. Proc Natl Acad Sci USA. 2001, 98: 13266-13271. 10.1073/pnas.221465998.PubMed CentralView ArticlePubMedGoogle Scholar
- Robbins J: Thyroid hormone transport proteins and the physiology of hormone binding. In The Thyroid. Edited by: Braverman LE, Utiger RD. 2000, Philadelphia, PA: Lippincott William and Wilkins, 105-120. 8Google Scholar
- Sotiriou C, Powles TJ, Dowsett M, Jazaeri AA, Feldman AL, Assersohn L, Gadisetti C, Libutti SK, Liu ET: Gene expression profiles derived from fine needle aspiration correlate with response to systemic chemotherapy in breast cancer. Breast Cancer Res. 2002, 4: R3-10.1186/bcr433.PubMed CentralView ArticlePubMedGoogle Scholar
- Sadow PM, Chassande O, Gauthier K, Samarut J, Xu J, O'Malley BW, Weiss RE: Specificity of thyroid hormone receptor subtype and steroid receptor coactivator-1 on thyroid hormone action. Am J Physiol Endocrinol Metab. 2003, 284: E36-E46.View ArticlePubMedGoogle Scholar
- Miller L, Park K, Guo Q, Alkharouf N, Malek R, Lee N, Liu E, Cheng SY: Silencing of Wnt signaling and activation of multiple metabolic pathways in response to thyroid hormone-stimulated cell proliferation. Mol Cell Biol. 2001, 21: 6626-6639. 10.1128/MCB.21.19.6626-6639.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Dillmann WH, Gloss BR: The role of thyroid hormone receptors in the heart. Methods Mol Biol. 2002, 202: 55-70. 10.1385/1-59259-174-4:55.PubMedGoogle Scholar
- Fadel BM, Ellahham S, Ringel MD, Lindsay J, Wartofsky L, Burman KD: Hyperthyroid heart disease. Clin Cardiol. 2000, 23: 402-408.View ArticlePubMedGoogle Scholar
- Refetoff S, Weiss RE, Usala SJ: The syndromes of resistance to thyroid hormone. Endocr Rev. 1993, 14: 348-399. 10.1210/er.14.3.348.PubMedGoogle Scholar
- Kahaly GJ, Matthews CH, Mohr-Kahaly S, Richards CA, Chatterjee VK: Cardiac involvement in thyroid hormone resistance. J Clin Endocrinol Metab. 2002, 87: 204-212. 10.1210/jc.87.1.204.View ArticlePubMedGoogle Scholar
- Gene Ontology Consortium. [http://www.geneontology.org]
- Reyne Y, Nougues J, Cambon B, Viguerie-Bascands N, Casteilla L: Expression of c-erbA alpha, c-erbA beta and Rev-erbA alpha mRNA during the conversion of brown adipose tissue into white adipose tissue. Mol Cell Endocrinol. 1996, 116: 59-65. 10.1016/0303-7207(95)03696-2.View ArticlePubMedGoogle Scholar
- Viguerie N, Millet L, Avizou S, Vidal H, Larrouy D, Langin D: Regulation of human adipocyte gene expression by thyroid hormone. J Clin Endocrinol Metab. 2002, 87: 630-634. 10.1210/jc.87.2.630.View ArticlePubMedGoogle Scholar
- Pucci E, Chiovato L, Pinchera A: Thyroid and lipid metabolism. Int J Obes Relat Metab Disord. 2000, S109-S112. 24 Suppl 2Google Scholar
- Huber O: Structure and function of desmosomal proteins and their role in development and disease. Cell Mol Life Sci. 2003, 60: 1872-1890. 10.1007/s00018-003-3050-7.View ArticlePubMedGoogle Scholar
- Koibuchi N, Chin WW: Thyroid hormone action and brain development. Trends Endocrinol Metab. 2000, 11: 123-128. 10.1016/S1043-2760(00)00238-1.View ArticlePubMedGoogle Scholar
- Poguet AL, Legrand C, Feng X, Yen PM, Meltzer P, Samarut J, Flamant F: Microarray analysis of knockout mice identifies cyclin D2 as a possible mediator for the action of thyroid hormone during the postnatal development of the cerebellum. Dev Biol. 2003, 254: 188-199. 10.1016/S0012-1606(02)00039-8.View ArticlePubMedGoogle Scholar
- Chanoine JP, Braverman LE: The role of transthyretin in the transport of thyroid hormone to cerebrospinal fluid and brain. Acta Med Austriaca. 1992, 19 Suppl 1: 25-28.PubMedGoogle Scholar
- Vincent J, Legrand C, Rabie A, Legrand J: Effects of thyroid hormone on synaptogenesis in the molecular layer of the developing rat cerebellum. J Physiol (Paris). 1982, 78: 729-738.Google Scholar
- Madeira MD, Paula-Barbosa MM: Reorganization of mossy fiber synapses in male and female hypothyroid rats: a stereological study. J Comp Neurol. 1993, 337: 334-352.View ArticlePubMedGoogle Scholar
- Hall AC, Lucas FR, Salinas PC: Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell. 2000, 100: 525-535. 10.1016/S0092-8674(00)80689-3.View ArticlePubMedGoogle Scholar
- Salinas PC: Wnt factors in axonal remodelling and synaptogenesis. Biochem Soc Symp. 1999, 65: 101-109.PubMedGoogle Scholar
- Feng X, Jiang Y, Meltzer P, Yen PM: Thyroid hormone regulation of hepatic genes in vivo detected by complementary DNA microarray. Mol Endocrinol. 2000, 14: 947-955. 10.1210/me.14.7.947.View ArticlePubMedGoogle Scholar
- Wood WM, Sarapura VD, Dowding JM, Woodmansee WW, Haakinson DJ, Gordon DF, Ridgway EC: Early gene expression changes preceding thyroid hormone-induced involution of a thyrotrope tumor. Endocrinology. 2002, 143: 347-359. 10.1210/en.143.2.347.View ArticlePubMedGoogle Scholar
- Davis PJ, Tillmann HC, Davis FB, Wehling M: Comparison of the mechanisms of nongenomic actions of thyroid hormone and steroid hormones. J Endocrinol Invest. 2002, 25: 377-388.View ArticlePubMedGoogle Scholar
- Shupnik MA, Chin WW, Habener JF, Ridgway EC: Transcriptional regulation of the thyrotropin subunit genes by thyroid hormone. J Biol Chem. 1985, 260: 2900-2903.PubMedGoogle Scholar
- Goodridge AG: Regulation of the gene for fatty acid synthase. Fed Proc. 1986, 45: 2399-2405.PubMedGoogle Scholar
- Gharbi-Chihi J, Facchinetti T, Berge-Lefranc JL, Bonne J, Torresani J: Triiodothyronine control of ATP-citrate lyase and malic enzyme during differentiation of a murine preadipocyte cell line. Horm Metab Res. 1991, 23: 423-427.View ArticlePubMedGoogle Scholar
- Sugisaki T, Noguchi T, Beamer WG, Kozak LP: Genetic hypothyroid mice: normal cerebellar morphology but altered glycerol-3-phosphate dehydrogenase in Bergmann glia. J Neurosci. 1991, 11: 2614-2621.PubMedGoogle Scholar
- Xiong S, Chirala SS, Hsu MH, Wakil SJ: Identification of thyroid hormone response elements in the human fatty acid synthase promoter. Proc Natl Acad Sci USA. 1998, 95: 12260-12265. 10.1073/pnas.95.21.12260.PubMed CentralView ArticlePubMedGoogle Scholar
- Weitzel JM, Kutz S, Radtke C, Grott S, Seitz HJ: Hormonal regulation of multiple promoters of the rat mitochondrial glycerol-3-phosphate dehydrogenase gene: identification of a complex hormone-response element in the ubiquitous promoter B. Eur J Biochem. 2001, 268: 4095-4103. 10.1046/j.1432-1327.2001.02332.x.View ArticlePubMedGoogle Scholar
- Liu YY, Schultz JJ, Brent GA: A thyroid hormone receptor alpha gene mutation (P398H) is associated with visceral adiposity and impaired catecholamine-stimulated lipolysis in mice. J Biol Chem. 2003, 278: 38913-38920. 10.1074/jbc.M306120200.View ArticlePubMedGoogle Scholar
- Barrera-Hernandez G, Park KS, Dace A, Zhan Q, Cheng SY: Thyroid hormone-induced cell proliferation in GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity. Endocrinology. 1999, 140: 5267-5274. 10.1210/en.140.11.5267.View ArticlePubMedGoogle Scholar
- Pibiri M, Ledda-Columbano GM, Cossu C, Simbula G, Menegazzi M, Shinozuka H, Columbano A: Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). FASEB J. 2001, 15: 1006-1013. 10.1096/fj.00-0416com.View ArticlePubMedGoogle Scholar
- Gonzalez-Sancho JM, Figueroa A, Lopez-Barahona M, Lopez E, Beug H, Munoz A: Inhibition of proliferation and expression of T1 and cyclin D1 genes by thyroid hormone in mammary epithelial cells. Mol Carcinog. 2002, 34: 25-34. 10.1002/mc.10046. (Erratum in: Mol Carcinog 2002, 34:164)View ArticlePubMedGoogle Scholar
- Perez-Juste G, Aranda A: The cyclin-dependent kinase inhibitor p27(Kip1) is involved in thyroid hormone-mediated neuronal differentiation. J Biol Chem. 1999, 274: 5026-5031. 10.1074/jbc.274.8.5026.View ArticlePubMedGoogle Scholar
- Nishikawa A, Kaiho M, Yoshizato K: Cell death in the anuran tadpole tail: thyroid hormone induces keratinization and tail-specific growth inhibition of epidermal cells. Dev Biol. 1989, 131: 337-344.View ArticlePubMedGoogle Scholar
- James RA, Sarapura VD, Bruns C, Raulf F, Dowding JM, Gordon DF, Wood WM, Ridgway EC: Thyroid hormone-induced expression of specific somatostatin receptor subtypes correlates with involution of the TtT-97 murine thyrotrope tumor. Endocrinology. 1997, 138: 719-724. 10.1210/en.138.2.719.PubMedGoogle Scholar
- Dinda S, Sanchez A, Moudgil V: Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells. Oncogene. 2002, 21: 761-768. 10.1038/sj.onc.1205136.View ArticlePubMedGoogle Scholar
- Kastan MB, Zhan Q, el-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, Fornace AJ: A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992, 71: 587-597.View ArticlePubMedGoogle Scholar
- el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B: WAF1, a potential mediator of p53 tumor suppression. Cell. 1993, 75: 817-825.View ArticlePubMedGoogle Scholar
- Jaiswal AS, Narayan S: p53-dependent transcriptional regulation of the APC promoter in colon cancer cells treated with DNA alkylating agents. J Biol Chem. 2001, 276: 18193-18199. 10.1074/jbc.M101298200.View ArticlePubMedGoogle Scholar
- Foster MP, Montecino-Rodriguez E, Dorshkind K: Proliferation of bone marrow pro-B cells is dependent on stimulation by the pituitary/thyroid axis. J Immunol. 1999, 163: 5883-5890.PubMedGoogle Scholar
- Watanabe K, Iwatani Y, Hidaka Y, Watanabe M, Amino N: Long-term effects of thyroid hormone on lymphocyte subsets in spleens and thymuses of mice. Endocr J. 1995, 42: 661-668.View ArticlePubMedGoogle Scholar
- Ruan H, Zarnowski MJ, Cushman SW, Lodish HF: Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes. J Biol Chem. 2003, 278: 47585-47593. 10.1074/jbc.M305257200.View ArticlePubMedGoogle Scholar
- Gloss B, Trost S, Bluhm W, Swanson E, Clark R, Winkfein R, Janzen K, Giles W, Chassande O, Samarut J, Dillmann W: Cardiac ion channel expression and contractile function in mice with deletion of thyroid hormone receptor alpha or beta. Endocrinology. 2001, 142: 544-550. 10.1210/en.142.2.544.PubMedGoogle Scholar
- Gloss B, Sayen MR, Trost SU, Bluhm WF, Meyer M, Swanson EA, Usala SJ, Dillmann WH: Altered cardiac phenotype in transgenic mice carrying the delta337 threonine thyroid hormone receptor beta mutant derived from the S family. Endocrinology. 1999, 140: 897-902. 10.1210/en.140.2.897.PubMedGoogle Scholar
- Taegtmeyer H, Cohen DM: Overestimating glycolysis in rat heart. Am J Physiol Endocrinol Metab. 2002, 283: E1102-1103. (Author reply E1103-1104)View ArticlePubMedGoogle Scholar
- Swanson EA, Gloss B, Belke DD, Kaneshige M, Cheng SY, Dillmann WH: Cardiac expression and function of thyroid hormone receptor beta and its PV mutant. Endocrinology. 2003, 144: 4820-4825. 10.1210/en.2003-0522.View ArticlePubMedGoogle Scholar
- Eisen MB, Brown PO: DNA arrays for analysis of gene expression. Methods Enzymol. 1999, 303: 179-205.View ArticlePubMedGoogle Scholar
- Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM: High-fidelity mRNA amplification for gene profiling. Nat Biotechnol. 2000, 18: 457-459. 10.1038/74546.View ArticlePubMedGoogle Scholar
- National Cancer Institute: Center for Cancer Research. [http://nciarray.nci.nih.gov]
- Genome Institute of Singapore: supplementary information. [http://www.gis.a-star.edu.sg/homepage/toolssup.jsp]
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text:
Hi Everyone,
Sorry I have been absent! Being such an IT dinosaur something changed on the iPad, so I haven't been able to blog.
I am back and guess what Gail B is getting for Christmas!!! We are getting a class room. A big classroom. Approval from the landlord has just come through, so the planning has just begun.
Keep checking in and I will show you some of the new ranges which have arrived over the last month or so.
Gail B
- Posted using BlogPress from my iPad

subset: commoncrawl


text:
Good info and nicely documented. Thanks for sharing Martin.
In reality dimensions in an InfoCube are often designed by business terms (like material, customer etc.) This often leads to the impression that InfoCube dimensions should be designed based on business constraints. This although should not be the leading criteria and shouldn't drive the decision.
Aside from the datavolume which depends on the granularity of the data in the InfoCube, performance is very much depending on how the InfoObject are arranged in the dimensions. Although this has no impact on the size of the fact table it certainly has one on the size of the dimensions.
How is a dimension then designed?
The main goal distributing the InfoObjects in their dimensions must be to keep the dimensions as small as possible. The decision on how many dimension and what InfoObjects go where is purely technical driven. In some cases this matches the organisational view but this would only be a conicidence and not the goal.
There is a few guidelines that should be considered assigning InfoObjects to dimensions:
- Use as many dimensions as necessary but it's more important to minimize dimension size rather than the number of dimensions.
- Within the dimension only characteristics that have a 1:n relation should be added (e.g. material and product hierarchy)
- Within a dimension there shouldn't be n:m relations. (e.g. product hierarchy and customer)
- Document level InfoObjects or big characteristics should be designed as Line-Item dimensions. Line item dimensions are not a true dimensions they have a direct link between the fact table and the SID table.
- The most selective characteristics should be at the top of the dimension table
- Don't mix characteristics with values that change frequently causing large dimension tables. (e.g. material and promotions)
- Consider also to combine unrelated characteristics it can improve performance by reducing the number of table joins. (you only have 13 dimensions so combine the small ones)
As a help the report (SE38) SAP_INFOCUBE_DESIGNS can be used.
This yellow marked dimension should be converted into a line item dimension if it contains a document level characteristic or it is simply bad design.
The maximum number of entries a dimension potentially can have is calculated through the cartesian product of all SID's. (e.g. 10'000 customer and 1'000 product hierarchies lead to 10'000'000 possible combinations in the dimension table. It's unlikely that this is going to happen and while designing the dimension this should also be considered – analyzing the possibilities of all customers buying all products in this case.
In cases where there is an m:m relationship it usually means there is a missing entity between those two and therefore they should be stored in different dimensions.
Once data is loaded into the InfoCube a check on the actual number of records loaded into the dimension table vs. the number of record in the fact table should be done. As a rule of thumb the ratio should be between 1:10 and 1:20.
Degenerated Dimensions
If a large dimension table reaches almost the size of the fact table when measured the number of rows in the tables it's a degenerated dimension. The OLAP processer has to join two big tables which is bad for the query perfromance. Such dimensions can be marked as Line Item Dimensions causing the database not to create an actual dimension table. Checking the table /BIC/F<INFOCUBE> will then show that instead of the DMID dimension key the SID of the degenerated dimension table is placed in the fact table. (Field name RSSID). With this a join of the two tables is eliminated. Those dimensions can only hold one InfoObject as a 1:1 relationship must exist between the SID value and the DIMID.
Dimensions with a lot of unique values can be set to High Cardinality which changes the method of indexing dimensions. (ORA DB only) This results in a switch from a bitmap index to a B-Tree index.
Defining a dimension as Line Item Dimension / High Cardinality
Finding the optimal model and balancing the size and the number of dimensions is a delicate excercise.
Dimensions in MultiProvider do not have to follow the underlying InfoCubes definitions. Those can be focused on the end users need and be structrured by the organizations meaning. This does not affect the performance as the MultiProvider does not have a physically existing datamodel on the database.
Designing the dimension in an InfoCube correctly can have a significant improvement on performance!
Hi Martin,
You really hit the nail on the head.with this block. You are blog as usual very informative and gives very practical tips.
Many thanks for sharing wonderful blog.
Anand Kumar


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text: I have a host (eth0 = 10.0.30.251) to which I recently added a virtual interface (eth0:0 = 10.0.30.249). Now all traffic that originates from this host has the source address of eth0:0, which is causing some problems with a firewall that I don't control. The output of both the route and netstat -rn commands shows eth0 as being the default interface. I'm doing all my interface configuration through the /etc/network/interfaces file and have not added any routes specifically for the eth0:0 interface (it was just added for a virtual web server). A friend has suggested that it is typical for an interface to choose the numerically lowest IP address that has been assigned to it as as the source address for outbound packets, but I haven't found anything to support that. Any help much appreciated! Matthew Whitworth


text:
Submitted by todd on
Cottonwood AZ Open Source Software Society (Cottonwood)
(google map) (yahoo map)
Hello. My name is Mike and I like Free and Open-Source Software (FOSS). I'm attempting to find a group of people who are like minded. Presently, I'm looking for people who would be interested in socializing but in the future I plan on trying to hold computer-club meetings. The purpose of these meetings is both to be social and to try to teach about open source software. I know there is a computer club here in the Verde Valley but they charge for membership. We will meet at coffee shops and other such places so, as Linux is free, the club will be as well. A third thing I would like to do is to periodically have, what they call in Phoenix, installfests. Installfests are events where people, who wish to test FOSS gather together to install a FOSS operating system on their computers (usually as a dual-boot so as to preserve their windows installation). Please, contact me and we'll get together :) Name of the group? Why of course.... it is COSSS.
feel free to contact Todd if youhave are interested a Cottonwood event
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text:
SOS Canal St.Posted by MJ
Same Old Situation (S.O.S) on Canal St. The Buffalo news reported another bidder has pulled out.
A clothing store has withdrawn its offer to buy a building at 51 Canal St. because its owner didn't feel the city was really interested in him.
Lindsey Hicks, of Picasso Moon, who wanted to relocate his business from Main Street, said last week he might end up in Buffalo.
His was the clothing business city officials previously said wanted 51 Canal St.
A recreational sales business wants to buy both 51 and 79-81 Canal St., while a third contender, a gift and decor shop interested only in 79-81 Canal, seems to have receded in the city's view
R. Charles Bell, city director of planning and development, said the recreational sales business may feature seasonal activity offerings of potential tourist interest, such as rentals of bicycles and other sporting goods.
However, he said, the deal isn't ready for the board of the city's development corporation to vote on yet, though that could change by meeting time Thursday..Once the the final establishments, if any, pop up on Canal St. we'll have to take a look back and see if all this drama was worth it. The street is so sparse on store fronts that regardless of what goes in there it will hardly be a place of abundant activity. The gazebo plot would need infill with upper residential of any hope of critical mass. As always, I'll always pray that we someday see the original Niagara Wine Trail concept reappear.
As I have mentioned before, it would also be great to see a Trek sign on the Canal facade of their building to show people driving by some type of life on the street.


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text:
via CAAI
Published: 5/02/2011
The deteriorating situation along the Thai-Cambodian border is undermining confidence in Asean and affecting economic recovery, tourism and investment prospect in the region, said Surin Pitsuwan, Secretary-General of the Association of Southeast Asian Nations in his urgent message to the two ASEAN Member States now trading fires with casualties on both sides.
"I am deeply concerned about the serious situation on the border between Thailand and Cambodia. This violent conflict must be brought under control and return to negotiating table soonest," said Dr. Surin.
The violent conflict started with gun fire and artillery duels mid afternoon Friday near the Preah Vihear temple. While the International Court of Justice decided in favor of Cambodia in 1962, the areas adjacent to the Temple remain under dispute.
"I have been in touch with Deputy Prime Minister and Minister for Foreign Affairs of Cambodia Hor Namhong, and the Foreign Minister of Thailand, Kasit Piromya, and I have appealed for calm, maximum restraint on both sides, and expressed my fervent desire to see both sides return to a negotiating table as soon as possible, said the Asean Secretary-General.
Dr Surin Pitsuwan also called both sides to allow Asean to help bring them to some form of a temporary truce and cool down the emotions and temper so that a higher interest of both peoples and that of Asean can be protected and enhanced.
"The situation has escalated into open conflict. And that will definitely affect our economic development, confidence in our region, and tourism and prospect for foreign investment, which have just been picking up in light of the world economic recovery," said Secretary-General Surin Pitsuwan, who is a former Foreign Minister of Thailand.
Diplomatic sources said Indonesia, the current chair of Asean, is also stepping up its diplomatic efforts to help the two sides arrive at a temporary solution so as to allow the bilateral mechanisms between them to accomplish their objectives of border demarcation and a general peace in the areas.
"I understand both sides now welcome some form of mediation by the Asean leadership," Surin said, without elaborating.


text:
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text:
What is the function of the festival passes?
The handed out passes are solely intended as a souvenir!
They represent no access authorization. Except the AAA-passes of authorizing staff.
Those can be identified by the shrink-wrapped photos of the pass holders.
Access authorization is defined by the individual wristband code.
What is the function of the festival passes?


text:
BofA Business Capital Provides E.C. Barton & Company Revolver
Bank of America Business Capital completed a $55 million senior secured revolving credit facility for longtime client E.C. Barton & Company to refinance the company's existing debt and provide ongoing working capital financing.
"E.C. Barton & Company is pleased to have entered into this financing agreement with Bank of America Merrill Lynch," said Niel Crowson, president and CEO of E.C. Barton & Company. "We have had a long and successful relationship with Bank of America Merrill Lynch, and this latest transaction exemplifies the commitment and trust we have for our financial provider. We value this collaborative relationship and look forward to continuing to grow together."
Wilson Moore, senior vice president in Global Commercial Banking at Bank of America Merrill Lynch, has managed the E.C. Barton relationship for more than 15 years. "We are excited to continue our rich and meaningful history with E.C. Barton & Company," Moore said. "It has been a pleasure to watch the company grow over the years, and we have been pleased to offer the financial solutions to help along the way".
Jeff McLane, president of Bank of America Business Capital, said, "We are delighted to provide this financing to help E.C Barton continue its growth. We place a high value on the relationship E.C. Barton has had with our firm, and we look forward to supporting the company in the coming years".
E. C. Barton & Company, a member of industry cooperative Do It Best, sells lumber and building materials through roughly 120 locations in the U.S.


text:
понеделник, 4 февруари 2013 г.
Good vs Bad part 3
40 коментара:
Thank you for your visit and i love the fotos. Great face painting.
see you soon
Nice make ups :X
I love you blog, would you like to follow each other?
- Анонимен4 февруари 2013 г., 14:47 ч.
Hi Veselina!!! I love Ursula look!! Follow me please!! Thanks!! Kisses!ОтговорИзтриване
wow that looks amazing!ОтговорИзтриване
♥ Ellen
Facebook + Instagram
You're very talented with makeup I'm jealous. Beautiful face art!!ОтговорИзтриване
I really like your blog, following you on GFC now.
Would love it if you checked out my blog! xx
Thank you for dropping by my blog and leaving me a comment!ОтговорИзтриване
Lovely pictures.
Yeah, I would like to follow each other. I am following you now both on bloglovin and GFC :)
xx, A!
This is a really cute post!ОтговорИзтриване
Your make up is done so well!ОтговорИзтриване
Abolutely love your work!ОтговорИзтриване
Lovely blog you got here doll, would love to follow each other!
Follow me on GFC and Facebook and i will follow you back;)
This is my Facebook page: https://www.facebook.com/pages/Juicy-Confashions/450396928311999?fref=ts
Love from Amsterdam!
After visiting your blog, I invite you to join us in the "International Directory Blogspot".
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Missing yours join us
If you join us and follow our blog, you will have many more visitors.
It's very simple, you just have to follow our blog, enter your Country and your blog url in a comment, and you will be automatically integrate into the Country list.
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After your approval to join us, you will receive your badge
We ask that you follow our blog and place a badge of your choice on your blog, in order to introduce the "directory" to your friends.
I follow your blog, I hope it will please you
To find out more about us, click on the link below:
I love your work dear. Sure we can follow each other. Thanks for commenting on my blog. Actually, I just followed you. I hope you can get back to my blog and follow me, too.ОтговорИзтриване
XX, IamJenniya
I also believe in "...and they live happily ever after".ОтговорИзтриване
Очарователна си! Имаш нов последовател :3
-Mademoiselle S.
- Анонимен5 февруари 2013 г., 17:37 ч.
Really cool blog, never seen anything like it!ОтговорИзтриване
Check out my blog, more posts this week:)
Melina xx
Много си креативна! Успех! :)ОтговорИзтриване
love this! would you like to follow each other? let me know!ОтговорИзтриване
beautiful!!! maybe we could follow each other via gfc/fb/bloglovin?! It would be a pleasure for me :)ОтговорИзтриване
Girl, you are so talented!! And your eyes are gorgeous!ОтговорИзтриване
I definitely love the idea of following each other xx
awesome make-up )ОтговорИзтриване
kisses )
Angela Donava
Hello from FranceОтговорИзтриване
I am very happy to welcome you!
Your blog has been accepted in Europe Bulgaria n°33 a minute!
Following our blog will gives you twice as many possibilities of visits to your blog!
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Invite your friends to join us in the "directory"!
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photography and poetry. So you will be able to find in different countries other people with passions similar to your ones.
We are fortunate to be on the Blogspot platform that offers the opportunity to speak to the world.
The more people will join, the more opportunities everyone will have. And yes, I confess, I need people to know this blog!
You are in some way the Ambassador of this blog in your Country.
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SO, you also have to make it known to your contacts and friends in your blog domain: the success of this blog depends on all Participants.
So, during your next comments with your friends, ask them to come in the 'Directory' by writing in your comments:
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You want this directory to become more important? Help me to make it grow up!
Your blog is in the list Europe Bulgaria n°33 and I hope this list will grow very quickly
We ask that you follow our blog and place a badge of your choice on your blog, in order to introduce the "directory" to your friends.
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Me encanta tu blog, pasaré por aquí a menudo. Se ve que trabajas mucho en él eso nu ca está mal obteniendo tan buenos resultados. Si quieres podemos seguirnos, pásate si te apeteceОтговорИзтриване
Puedes seguirme también en mi nuevo twitter @FATLAGERFF_
Un beso guapa!
I love it!nice blog. What about following each other?ОтговорИзтриване
- Анонимен6 февруари 2013 г., 11:51 ч.
So creative! OXОтговорИзтриване
I'm looking for new blogs to follow and love yours, want to follow each other? Let me know.
Hello! :) I love your photography and blog ! I am following. I hope you can check out my site: http://theproverbs.net and FOLLOW. Also on bloglovin, facebook, instagram, twitter, tumblr and even lookbook if you want :3 <3ОтговорИзтриване
Following you back on GFC doll,ОтговорИзтриване
lovely makeup!ОтговорИзтриване
from helen at thelovecatsinc // youtube
ps. enter my new giveaway to win the UD naked basics palette, real techniques brushes and essie polishes! click here.
super cool ;)ОтговорИзтриване
nice blog
follow back, if you like ;)
You have great Ideas and a interesting blog, i really like it!ОтговорИзтриване
what you think, wanna follow each other?
I would be happy :-) Kisses Kira from Switzerland
great post =)ОтговорИзтриване
glad that Ive come across your blog =)
come and see mine too =)
and we can follow each other via GFC and Bloglovin ^^
This is something amazing!!!! Great pictures! I'm following you right now, hope you will do same!ОтговорИзтриване
Have a fabulous time!
Best wishes, Alexandra
That's some lovely make-up art!ОтговорИзтриване
I'd like to see more of that! Therefor I'm now following your blog (:
Follow me back?
x Elena
Nice blog you have...wanna follow each other?ОтговорИзтриване
please follow with GFC, just leave me a comment and I'll follow u back!
Followed back!ОтговорИзтриване
Please check also my facebook page and like it if u can!
- Анонимен9 февруари 2013 г., 13:22 ч.
Really great makeup!ОтговорИзтриване
I'm hosting a give-away on my blog, maybe you're interested? check it out http://lemizysconfessionbook.blogspot.nl/2013/02/time-for-giveaway.html
following back! ^^ОтговорИзтриване
keep in touch^^
beautiful looks *_* your blog is very lovely and interesting. im glad if you visit my blog, too <3 keep in touch!ОтговорИзтриване
Great blog! I'm a new follower =)ОтговорИзтриване
its amazing! :)) good work :)ОтговорИзтриване


text:
Isle of Skye was bought when Caroline and I were going on vacation a couple of months ago. I got this idea that we should buy a couple of small games to bring with us to play at the hotel in the evenings or if the weather would turn bad.
This was the game that I
liked the most, and which also had the most "game" in it - surprisingly
how hard it is to find good small scale games...
Isle of Skye has a
resemblance to Carcasonne, a game I dislike. It's not the first time I like a
game similar to another that I don't however. In this case I found that the
artwork was better looking and the game less fiddly, and it also had a bit more
player interaction which brought the similar experience above average to good.
Isle of Skye is a tile placement
game, with an interesting bidding/money system and interchangeable objectives
to keep the game fresh.
Each player takes on the
role of a highland clan trying to build a kingdom using tiles. Points are
scored for a variety of objectives, such as most watchtowers, ships, hamlets,
cattle, beer etc.
It is in many senses
similar to Carcassonne in this regard, however the difference is that in Isle
of Skye all players have their own individual maps instead of a shared one. And
also, instead of randomly picking and placing a tile like you do in
Carcassonne, you pick 3 tiles per player each turn. After picking tiles you
place them in front of your player sheet - and behind your sheet you do a
secret bid.
The bid is how much you are willing to purchase each tile for, one of the three
tiles also needs to be scrapped.
As all players to this step
at the same time you first see what everyone has drafted, and plan how to
incorporate the tiles that other players drafted into your own kingdom. During
this step you also need to carefully plan your economy - do you save money for
purchase of opposite player tiles or do you really want to keep the tiles that
you drafted yourself and make them expensive to avert purchase?
As players reveal which
tiles are scrapped and how they have priced the remaining two tiles, players
take a round of bidding and purchasing. Starting with the first player all
players have the opportunity to bid on one tile located in front of an
opponent, or pass. You are only allowed to buy one tile per round. Smart
players will know what victory points their opponent aim for and price the
desired tiles expensive.
Should you pass during the
bidding step you are not allowed to buy anything this round. After the
bidding/purchase all players now place the remaining and purchased tiles that
they have on their map.
Tiles need to match in
terms of geographic features, roads must not connect but oftentimes there are
additional victory points to be made for linking resources by roads to
your castle.
Isle of Skye is a simple
but elegant boardgame that provides opportunities for tactical gameplay and
planning alike. With a multitude of victory conditions the game will remain
fresh as scoring elements alter in between games. Points are also scored in a
irregular pattern, which has you plan for both short and long term purchase and
placement of tiles as no single turn provides victory points for all 4 victory
conditions at the same time.
We have played this game
with 2-3 players and it works very well. I would recommend to play it with at
least 3 players, and hope to get a 4 player game sometime. Isle of Skye is a
perfect lightweight game with good boardgame depth to play with people that are
not regular boardgamers, and as I would imagine a good family game. Quick
rules, quick gameplay and you can manage several sessions during a single
afternoon with no problem.
It's hard to me to imagine
breaking this open with my regular boardgame group as we tend to play highly
thematic and long boardgames when we do get the opportunity to meet and play one afternoon. But if we did it would be part of a boardgame afternoon with a mix of small/fast boardgames such as 7 Wonders and the like.
Isle of Skye 7,5 / 10
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26 April 2017
Isle of Skye boardgame review
Posted by Anatoli at 08:26
Labels: Boardgame review
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text:
While a gurgling baby is enough to make some feel broody, others know that becoming a parent is simply not for them.
On Reddit, men and women have been sharing the moment they realised they didn't ever want to have kids.
From walking past a playground to being stuck in a car with a younger sibling, here are just a few of their stories.
1. "Walked past the 'play area' in a mall. The sound was deafening."
2. "When I had to ride in a car for 45 minutes with my little sister and a five-year old she was babysitting. For the entire ride, the kid screamed, pausing only to take a breath or giggle. It took 10 years, but I kept the promise I made that day and got sterilised. No regrets."
3. "When I was doing one of those 'what do you want to be when you grow up' things in school and realised, after someone asked why, that I never factored kids in there. As a girl, that's totally what you're supposed to do, right?
"I never could come up with an answer as to why I never factored having kids into that plan because, simply enough, I didn't want them. I was about 10 then, and I'm 26 now - still don't want kids and don't think I'll ever change my mind."
4. "When I watched an actual video of childbirth... Something beautiful sure looks gruesome."
5. "Every single time I walk by an obviously pregnant person. I never want my body to do that. Hard nope."
6. "I worked very hard to lose a lot of weight, kept it off for years, and finally got some control over my body image issues. The last thing I want is to have to go through all that again just to produce a screaming child that will probably wind up hating me anyway."
- delmar42
7. "When my mother, who is in obstetrics/gynaecology, sat me down at 18 and told me that having children was the worst decision she ever made.
"I am a lot like her and I could see myself repeating the same patterns of behaviour that negatively affected my own life with whatever spawn I might have. I actually really respect her for being honest with me and not perpetuating the 'having children is your purpose and a miracle' fallacy."
8. "In the eighth grade I had to take care of a robot baby. The first night I had it the damn thing woke me up at 2:00am screaming as loud as it could. My mum hears this and comes into my room. All she sees is me cradling this robot, not knowing what to do and freaking out (basically crying).
"My mum takes the baby into her room and starts cradling it until she thinks to herself 'wtf am I doing?' She then proceeds to wrap the baby in a series of blankets and put it in her closet for the rest of the night.
"I myself would proceed to do this every night until the assignment was over. Fuck that robot. That was eight-nine years ago and I still don't want any kids."
9. "Sounds terrible but my nephew made me realise I wan to stay child free. I know he was a baby but he was the most pitiful kid ever. He'd cry over anything.
"I don't have the patience for that and never will. Everyone says 'when it's your kid it's different'. No, it'll still be annoying and there won't be a 'my kid'."
10. "My girlfriend and I have chosen to not have children due to mental disorders on both sides of our family. We plan on having fur babies! We already have a hedgehog, his name is Kraken and a Chinchilla named Kthulu."
11. "I just looked around and felt that the world already had a sufficiency of children. Why add another one when we are already well over quota for population density in my area?
"Also, we don't make enough money to support a child the way I would like to. Growing up poor was not pleasant for me and I wouldn't want to make someone else do it. I'd rather save up and chip in for another promising kid's college who doesn't have the money than spend all that money doing half-assed financial stability on my own personal child."
12. "My mother decided to have one when I was 13. We lived in a small two bedroom apartment. They put his crib in their room (thank God) but on the same wall where the top of my bed was. I could not sleep for about nine months.
"I ended up having to restart my school grade. And I could never have friends over because it was too loud for the baby. So yeah, fuck the little bastards."
Sacbastein Thomas - Sth Photography via Getty Image
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text:
God is up to something. We humans have made a mess of our lives and God's creation, but God is at work redeeming what we've tried to ruin. That project of redemption is the driving force of human history. God doesn't need us for his project, but, by his grace, he invites us to participate. Ministry is everything we do to participate in that project.
At EPCC, we believe that people can't truly transform other people, only God can (John 15:4-8; Gal. 5:16-24). So, our ministry isn't about causing growth, it's about creating opportunity. If you're interested in drawing close to God and growing spiritually, we see it as our responsibility to do everything we can to equip you for that journey (Eph. 4:11-12). Please explore this section of our site to find out how we plan to achieve that goal.


text:
I had a misunderstanding with a very good friend of mine. I was thinking one thing and she was thinking another thing.
Well I gave myself a week to chill out so I wouldn't say something I would regret. During that week my friend built the misunderstanding into something bigger.
When we finally did get together to talk things out, I thought everything was OK but she wasn't so sure despite what I said.
I am hoping this card says it all and we can both be in a good place again.