"Role of long non-coing RNAs in the stressed heart". Wisper : a novel cardiac fibroblast-enriched IncRNA that modulate fibrosis in the failing heart

LncRNAs are transcripts greater than 200 nucleotides in length with no apparent coding potential. They exert important regulatory functions in the genome. Their role in cardiac fibrosis is however unexplored. To identify IncRNAs that could modulate cardiac fibrosis, we profiled the long non-coding transcriptome in the infarcted mouse heart, and identified 1500 novel IncRNAs. These IncRNAs have unique characteristics such as high tissue and cell type specificity. Their expression is highly correlated with parameters of cardiac dimensions and function. The majority of these novel IncRNAs are conserved in human. Importantly, human IncRNAs appear to be differentially expressed in heart disease. Using a computational pipeline, we identified a super-enhancer-associated IncRNA, which is dynamically expressed after myocardial infarction. We named this particular transcript Wisper for «Wisp2 super-enhancer- derived IncRNA ». Interestingly, Wisper expression is overexpressed in cardiac fibroblasts as compared to cardiomyocytes or to fibroblasts isolated from other organs than the heart. The importance of Wisper in the biology of fibroblasts was demonstrated in knockdown experiments. Differentiation of cardiac fibroblast into myofibroblasts in vitro is significantly impaired upon Wisper knockdown. Wisper downregulation in cardiac fibroblasts results in a dramatic reduction of fibrotic gene expression, a diminished cell proliferation and an increase in apoptotic cell death. In vivo, depletion of Wisper during the acute phase of the response to infarction is detrimental via increasing the risk of cardiac rupture. On the other hand, Wisper knockdown following infarction in a prevention study reduces fibrosis and preserves cardiac function. Since WISPER is detectable in the human heart, where it is associated with severe cardiac fibrosis, these data suggest that Wisper could represent a novel therapeutic target for limiting the extent of the fibrotic response in the heart. -- Les long ARN non-codants (IncRNAs) sont des ARN de plus de 200 nucléotides qui ne codent pas pour des protéines. Ils exercent d'importantes fonctions dans le génome. Par contre, leur importance dans le développement de la fibrose cardiaque n'a pas été étudiée. Pour identifier des IncRNAs jouant un rôle dans ce processus, le transcriptome non-codant a été étudié dans le coeur de'souris après un infarctus du myocarde. Nous avons découverts 1500 nouveaux IncRNAs. Ces transcrits ont d'uniques caractéristiques. En particulier ils sont extrêmement spécifiques de sous-populations de cellules cardiaques. Par ailleurs, leur expression est remarquablement corrélée avec les paramètres définissant les dimensions du coeur et la fonction cardiaque. La majorité de ces IncRNAs sont conservés chez l'humain. Certains sont modulés dans des pathologies cardiaques. En utilisant une approche bioinformatique, nous avons identifié un IncRNA qui est associé à des séquences amplificatrices et qui est particulièrement enrichi dans les fibroblastes cardiaques. Ce transcrit a été nommé Wisper pour «Wisp2 super-enhancer-derived IncRNA ». L'importance de Wisper dans la biologie des fibroblastes cardiaques est démontrée dans des expériences de déplétion. En l'absence de Wisper, l'expression de protéines impliquées dans le développement de la fibrose est dramatiquement réduite dans les fibroblastes cardiaques. Ceux-ci montrent une prolifération réduite. Le niveau d'apoptose est largement augmenté. In vivo, la déplétion de Wisper pendant la phase aiguë de l'infarctus rehausse le risque de rupture cardiaque. Au contraire, la réduction de l'expression de Wisper pendant la phase chronique diminue la fibrose cardiaque et améliore la fonction du coeur. Puisque Wisper est exprimé dans le coeur humain, ce transcrit représente une nouvelle cible thérapeutique pour limiter la réponse fibrotique dans le coeur.

IMP2 provides an alternative to LIN28B for LET-7 target gene protection and glioma stem cell maintenance

Glioblastoma multiforme (GBM) is the most frequent and lethal primary brain tumor in adults. Accumulating evidence suggests that tumors comprise a hierarchical organization that is, at least partially, not genetically driven. Cells that reside at the apex of this hierarchy are commonly referred to as cancer stem cells (CSCs) and are believed to largely contribute to recurrence and therapeutic failure. Although the complexity of epigenetic regulation of the genome precludes prediction as to which epigenetic changes dominate CSC specification in different cancer types, the ability of microRNAs (miRNAs) to fine-tune expression of entire gene networks places them among prime candidates for establishing CSC properties. In this study we characterized the miRNA expression profile of primary GBM grown either under conditions that enrich for GSCs or their differentiated non-tumorigenic progeny (DGCs). Although, we identified a subset of miRNAs that was strongly differentially expressed between GSCs and DGCs, we observed that in GSCs both let-7 and, paradoxically, their target genes are highly expressed, suggesting protection against let-7 action. Using PAR-CLIP we show that insulin-like growth factor-2 mRNA-binding protein 2 (IMP2) provides a mechanism for let-7 target gene protection that represents an alternative to LIN28A/B, which abrogates let-7 biogenesis in normal embryonic and certain malignant stem cells. By direct binding to miRNA recognition elements, IMP2 protects its targets from let-7 mediated decay. Importantly, depletion of IMP2 in GSCs strongly impairs their self- renewal properties and tumorigenicity in vivo, a phenotype that can be rescued by expression of LIN28B, suggesting that IMP2 mainly contributes to GSC maintenance by protecting let-7 target genes from silencing. Using mouse models, we show that depletion of IMP2 in neural stem cells (NSCs) induces let-7 target gene down-regulation, impairs their clonogenic capacity, and affects differentiation. Taken together, our observations describe a novel regulatory function of IMP2 in the let-7 axis whereby it supports GSC and NSC specification. Résumé (Français) Le glioblastome (GBM) est la tumeur primaire maligne du cerveau la plus fréquente. De nombreuses études ont démontré l'existence d'une organisation hiérarchique des cellules cancéreuses liée à des mécanismes épigénétiques. Les cellules qui se trouvent au sommet de cette hiérarchie sont appelées cellules souches cancéreuses (CSC), et contribuent à l'échec thérapeutique. Bien que la complexité des régulateurs épigénétiques permette difficilement de prédire quel mécanisme contribue le plus aux propriétés des CSC, la capacité des microRNAs (miRNAs) de réguler des réseaux entiers de gènes, les placent comme des candidats de premiers choix. Ici, nous avons caractérisé le profil d'expression des miRNAs dans des tumeurs primaires de GBM cultivées dans des conditions qui enrichissent soit pour les CSC, soit pour leur contrepartie de cellules cancéreuses différences (CCD). De manière surprenante et paradoxale la famille de miRNA let-7 et leurs gènes cibles étaient hautement exprimés dans les CSC, suggérant un mécanisme de protection contre l'action des let-7. Avec l'aide de la technologie PAR-CLIP, nous démontrons que la protéine IMP2, protège les mRNAs de l'action des let-7 et représente une alternative à Lin28A/B, qui d'ordinaire réprime fortement la maturation des let-7 dans les cellules souches embryonnaires et divers cancers. En se liant à la région ciblée par les let-7, IMP2 protège ses transcrits de l'action de cette classe de microRNA qui est tumoro-supressive. La déplétion d'IMP2 dans des CSC de GBM réduit fortement leur clonogénicité in vitro et leur tumorigénicité in vivo. Ceci peut être reversé en introduisant Lin28B dans des CSC de GBM, suggérant qu'IMP2 exerce ses fonctions pro-tumorigéniques en modulant l'axe let-7. Avec l'aide de modèles murins, nous observons que la déplétion de IMP2 dans les cellules souches neurales (CSN) induit une baisse de leur clonogénicité et des cibles des miRNAs let-7, suggérant une conservation de ce mécanisme entre les CSC de GBM et les CSN. En résumé, nos observations définissent une nouvelle fonction de IMP2 dans l'axe let-7 par lequel il contribue au maintien des propriétés des CSC et des CSN.

Epigenetic alterations in hippocampus of SAMP8 senescent mice and modulation by voluntary physical exercise.

The senescence-accelerated SAMP8 mouse model displays features of cognitive decline and Alzheimer's disease. With the purpose of identifying potential epigenetic markers involved in aging and neurodegeneration, here we analyzed the expression of 84 mature miRNAs, the expression of histone-acetylation regulatory genes and the global histone acetylation in the hippocampus of 8-month-old SAMP8 mice, using SAMR1 mice as control. We also examined the modulation of these parameters by 8 weeks of voluntary exercise. Twenty-one miRNAs were differentially expressed between sedentary SAMP8 and SAMR1 mice and seven miRNAs were responsive to exercise in both strains. SAMP8 mice showed alterations in genes involved in protein acetylation homeostasis such as Sirt1 and Hdac6 and modulation of Hdac3 and Hdac5 gene expression by exercise. Global histone H3 acetylation levels were reduced in SAMP8 compared with SAMR1 mice and reached control levels in response to exercise. In sum, data presented here provide new candidate epigenetic markers for aging and neurodegeneration and suggest that exercise training may prevent or delay some epigenetic alterations associated with accelerated aging.

Epigenetic alterations in hippocampus of SAMP8 senescent mice and modulation by voluntary physical exercise.

The senescence-accelerated SAMP8 mouse model displays features of cognitive decline and Alzheimer's disease. With the purpose of identifying potential epigenetic markers involved in aging and neurodegeneration, here we analyzed the expression of 84 mature miRNAs, the expression of histone-acetylation regulatory genes and the global histone acetylation in the hippocampus of 8-month-old SAMP8 mice, using SAMR1 mice as control. We also examined the modulation of these parameters by 8 weeks of voluntary exercise. Twenty-one miRNAs were differentially expressed between sedentary SAMP8 and SAMR1 mice and seven miRNAs were responsive to exercise in both strains. SAMP8 mice showed alterations in genes involved in protein acetylation homeostasis such as Sirt1 and Hdac6 and modulation of Hdac3 and Hdac5 gene expression by exercise. Global histone H3 acetylation levels were reduced in SAMP8 compared with SAMR1 mice and reached control levels in response to exercise. In sum, data presented here provide new candidate epigenetic markers for aging and neurodegeneration and suggest that exercise training may prevent or delay some epigenetic alterations associated with accelerated aging.

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes.

Interleukin-4 induced leukocyte differentiation

Monocytes, macrophages and dendritic cells (DCs) are important mediators of innate immune system, whereas T lymphocytes are the effector cells of adaptive immune responses. DCs play a crucial role in bridging innate and adaptive immunity. Naïve CD4+ Th progenitors (Thp) differentiate to functionally distinct effector T cell subsets including Th1, Th2 and Th17 cells, which while being responsible for specific immune functions have also been implicated in pathological responses, such as autoimmunity, asthma and allergy. The main objective of this thesis is to dissect the signalling networks involved in the IL-4 induced differentiation of two important leukocyte subtypes, Th2 cells and DCs. Gene expression profiling lead to identification of over 200 genes which are differentially expressed during cytokine induced differentiation of human monocytes to DCs or macrophages and which are likely to be essential for the proper biological functions of these cell types. Transcriptome analysis demonstrated the dynamic regulation of gene expression by IL-12 and IL-4 during the initiation of Th cell differentiation, which was partly counteracted by an immunosuppressive cytokine, TGFβ, present in the culture media. Results from RNAi mediated gene knockdown experiments and global gene expression analysis elucidated that SATB1 regulates multiple genes important for Th cell polarization or function as well as may compete with GATA3 for the reciprocal regulation of IL-5 transcription. In conclusion, the results obtained have extended our system-level understanding of the immune cell differentiation processes and provide an excellent basis for the further functional studies which could lead to development of improved therapeutic approaches for a range of immunological conditions.

Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups.METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method.RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol).CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

MicroRNAs as novel regulators of skeletal homeostasis

The human skeleton is composed of bone and cartilage. The differentiation of bone and cartilage cells from their bone marrow progenitors is regulated by an intrinsic network of intracellular and extracellular signaling molecules. In addition, cells coordinate their differentiation and function through reciprocal cell‐to‐cell interactions. MicroRNAs (miRNAs) are small, single‐stranded RNA molecules that inhibit protein translation by binding to messenger RNAs (mRNAs). Recent evidence demonstrates the involvement of miRNAs in multiple biological processes. However, their role in skeletal development and bone remodeling is still poorly understood. The aim of this thesis was to elucidate miRNA‐mediated gene regulation in bone and cartilage cells, namely in osteoblasts, osteoclasts, chondrocytes and bone marrow adipocytes. Comparison of miRNA expression during osteogenic and chondrogenic differentiation of bone marrow‐derived mesenchymal stem cells (MSCs) revealed several miRNAs with substantial difference between bone and cartilage cells. These miRNAs were predicted to target genes essentially involved in MSC differentiation. Three miRNAs, miR‐96, miR‐124 and miR‐199a, showed marked upregulation upon osteogenic, chondrogenic or adipogenic differentiation. Based on functional studies, these miRNAs regulate gene expression in MSCs and may thereby play a role in the commitment and/or differentiation of MSCs. Characterization of miRNA expression during osteoclastogenesis of mouse bone marrow cells revealed a unique expression pattern for several miRNAs. Potential targets of the differentially expressed miRNAs included many molecules essentially involved in osteoclast differentiation. These results provide novel insights into the expression and function of miRNAs during the differentiation of bone and cartilage cells. This information may be useful for the development of novel stem cell‐based treatments for skeletal defects and diseases.

Regulation of lymphocyte response in vitro and in vivo

CD4+ T helper (Th) cells have an important role in the defence against diverse pathogens. Th cells can differentiate into several functionally distinct subtypes including Th1 and Th2 cells. Th1 cells are important for eradicating intracellular pathogens, whereas Th2 cells pro¬tect our body against extracellular parasites. However if uncontrolled, Th cells can mediate immunopathology such as asthma or allergies, but inappropriate Th response can also lead to autoimmune diseases such as multiple sclerosis or type 1 diabetes. Deeper knowledge of the regulation of the lymphocyte response both in vitro and in vivo is important for un¬derstanding the pathogenesis of immune-mediated diseases and microbe-host interactions. In the work presented in this thesis, the first goal was to elucidate the role of novel factors, PIM kinases and c-FLIP in the regulation of human Th cell differentiation. The oncogenic serine-threonine kinases of the PIM family were shown to be preferentially expressed in Th1 cells and in addition, by using RNA interference, they were also shown to be positive regulators of Th1 differentiation. The PIM depletion experiments suggest that PIM kinases promote the expression of the hallmark cytokine of Th1 cells, IFNγ, and influence the IL12/STAT4 pathway during the early Th1 cell differentiation. In addition to cytokine and T cell receptor (TCR) induced pathways, caspase activity has been shown to regulate Th cell proliferation. In the work presented in this thesis, the two isoforms of the caspase regulator protein, c-FLIP, were shown to be differentially ex¬pressed in Th1 and Th2 cells. Both of the isoforms were up-regulated in response to TCR activation, but the expression of the short isoform was selectively induced by IL4, the Th2 inducing cytokine. Furthermore, the c-FLIP isoforms had distinct and opposite roles during the early differentiation of Th1 and Th2 cells. The knockdown of the long isoform of c-FLIP led to the induction of Th1 marker genes, such as IFNγ and TBET, whereas the depletion of c-FLIP short down-regulated Th2 marker genes IL-4 and GATA3. The third goal was to elucidate the gene expression profiles characterizing the T- and B-lymphocyte responses in vivo during experimental infection by intracellular bacte¬rium Chlamydia pneumoniae. Previously, it has been shown that CD8+ and CD4+ T cells are important for the protection against Chlamydia pneumoniae. In this study, the analysis revealed up-regulation of interferon induced genes during recurrent infection underlining the importance of IFNγ secreted by Th1 and CD8+ T cells in the protection against this pathogen. Taken together, in this study novel regulators of Th cell differ¬entiation were discovered and in addition the gene expression profiles of lymphocytes induced by Chlamydia pneumoniae infection were characterized.

Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

Formin proteins in normal tissues and cancer

The actin cytoskeleton is a dynamic structure that determines cell shape. Actin turnover is mandatory for migration in normal and malignant cells. In epithelial cancers invasion is frequently accompanied by epithelial to mesenchymal transition (EMT). In EMT, cancer cells acquire a migratory phenotype through transcriptional reprogramming. EMT requires substantial re-organization of actin. During the past decade, new actin regulating proteins have been discovered. Among these are members of the formin family. To study formin expression in tissues and cells, antibodies for detection of formin proteins FMNL1 (Formin-like protein 1), FMNL2 (Formin-like protein 2) and FHOD1 (Formin homology 2 domain containing protein 1) were used. The expression of formins was characterized in normal tissues and selected cancers using immunohistochemistry. The functional roles of formins were studied in cancer cell lines. We found that FMNL2 is widely expressed. It is a filopodial component in cultured melanoma cells. In clinical melanoma, FMNL2 expression has prognostic significance. FHOD1 is a formin expressed in mesenchymal cell types. FHOD1 expression is increased in oral squamous cell carcinoma (SCC) EMT. Importantly, FHOD1 participates in invasion of cultured oral SCC cells. FMNL1 expression is low in normal epithelia, but high in leukocytes and smooth muscle cells. Expression of FMNL1 can be found in carcinoma; we detected FMNL1 expressing cells in basal type of breast cancer. Our results indicate that formins are differentially expressed in normal tissues and that their expression may shift in cancer. Functionally FMNL2 and FHOD1 participate in processes related to cancer progression. Studying formins is increasingly important since they are potential drug targets.

High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK), while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Pipeline for macro- and microarray analyses

The pipeline for macro- and microarray analyses (PMmA) is a set of scripts with a web interface developed to analyze DNA array data generated by array image quantification software. PMmA is designed for use with single- or double-color array data and to work as a pipeline in five classes (data format, normalization, data analysis, clustering, and array maps). It can also be used as a plugin in the BioArray Software Environment, an open-source database for array analysis, or used in a local version of the web service. All scripts in PMmA were developed in the PERL programming language and statistical analysis functions were implemented in the R statistical language. Consequently, our package is a platform-independent software. Our algorithms can correctly select almost 90% of the differentially expressed genes, showing a superior performance compared to other methods of analysis. The pipeline software has been applied to 1536 expressed sequence tags macroarray public data of sugarcane exposed to cold for 3 to 48 h. PMmA identified thirty cold-responsive genes previously unidentified in this public dataset. Fourteen genes were up-regulated, two had a variable expression and the other fourteen were down-regulated in the treatments. These new findings certainly were a consequence of using a superior statistical analysis approach, since the original study did not take into account the dependence of data variability on the average signal intensity of each gene. The web interface, supplementary information, and the package source code are available, free, to non-commercial users at http://ipe.cbmeg.unicamp.br/pub/PMmA.