Isolamentos e caracterização de DETs(Differentially Expressed Transcripts) em espécies de peixes: Leporinus macrocephalus(Characiformes) e Danio rerio (Cypriniformes)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serviço social, educação e complexidade: um diálogo com o homo complexus professor

Pós-graduação em Serviço Social - FCHS

Angiotensin II AT(1) receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT, receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2(+) influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation. (c) 2005 Elsevier B.V. All rights reserved.

cDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin

Dermatophytes are adapted to infect skin, hair and nails by their ability to utilize keratin as a nutrient source. Trichophyton rubrum is an anthropophilic fungus, causing up to 90% of chronic cases of dermatophytosis. The understanding of the complex interactions between the fungus and its host should include the identification of genes expressed during infection. To identify the genes involved in the infection process, representational difference analysis (RDA) was applied to two cDNA populations from T. rubrum, one transcribed from the RNA of fungus cultured in the presence of keratin and the other from RNA generated during fungal growth in minimal medium. The analysis identified differentially expressed transcripts. Genes related to signal transduction, membrane protein, oxidative stress response, and some putative virulence factors were up-regulated during the contact of the fungus with keratin. The expression patterns of these genes were also verified by real-time PCR, in conidia of T. rubrum infecting primarily cultured human keratinocytes in vitro, revealing their potential role in the infective process. A better understanding of this interaction will contribute significantly to our knowledge of the process of dermatophyte infection.

Transcriptionally active LTR retrotransposons in Eucalyptus genus are differentially expressed and insertionally polymorphic

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genes differentially expressed by Mycobacterium tuberculosis after exposure to ruthenium phosphinic compound and isoniazid

Background- The evaluation of the effects of new compounds and nonconventional anti-tuberculous drugs have grown and become increas-ingly more popular in recent years. Studies have shown anti-tuberculous activity for Ruthenium complexes, including organometallic com-pounds containing phosphine ligands such as picolinic acid generating great expectations and hopes. Methods- The Representational Difference Analysis (RDA) was applied in order to gain insight about differences in expression of Mycobacte-rium tuberculosis H37Rv exposed to [Ru(dppb)(pic)(bypy)] PF6 (SCAR1) and isoniazid (INH). Total RNA was extracted from the bacillus not exposed and exposed to SCAR1 and INH separately at concentration of MIC for 12 hours at 35°C. RDA was carried out and differentially expressed products were sequenced. Results- RDA-sequencing identified, for both compounds, orthologs that encode hypothetical and predict proteins. One related cell wall syn-thesis gene, identified by RDA, and genes related to INH target as inhA, katG and ahpC had their expression confirmed and quantified by real-time PCR. The gene encoding the cell wall associated hydrolase was induced 4.627 and 1.189, inhA 0.983 and 1.027, katG 1.111 and 1.345 and ahpC 1.063 and 1.039 fold after exposure to SCAR1 and INH respectively, compared to not exposed growth. Conclusion- The RDA brings, for the first time, directions to study related genes with metabolic pathways of SCAR1. RDA and Real-Time PCR highlight the idea that one of the SCAR1 interaction, in M tuberculosis may be in the cell wall biosynthesis considering the differential expression of a cell wall hydrolase and warrants further investigation.

Sexual Risk Recognition Deficits: The Role of Prior Victimization and Emotion Dysregulation

Recent theoretical writings suggest that the ineffective regulation of negative emotional states may reduce the ability of women to detect and respond effectively to situational and interpersonal factors that increase risk for sexual assault. However, little empirical research has explored this hypothesis. In the present study, it was hypothesized that prior sexual victimization and negative mood state would each independently predict poor risk recognition and less effective defensive actions in response to an analogue sexual assault vignette. Further, these variables were expected to interact to produce particularly impaired risk responses. Finally, that the in vivo emotion regulation strategy of suppression and corresponding cognitive resource usage (operationalized as memory impairment for the vignette) were hypothesized to mediate these associations. Participants were 668 female undergraduate students who were randomly assigned to receive a negative or neutral film mood induction followed by an audiotaped dating interaction during which they were instructed to indicate when the man had “gone too far” and describe an adaptive response to the situation. Approximately 33.5% of the sample reported a single victimization and 10% reported revictimization. Hypotheses were largely unsupported as sexual victimization history, mood condition, and their interaction did not impact risk recognition or adaptive responding. However, in vivo emotional suppression and cognitive resource usage were shown to predict delayed risk recognition only. Findings suggest that contrary to hypotheses, negative mood (as induced here) may not relate to risk recognition and response impairments. However, it may be important for victimization prevention programs that focus on risk perception to address possible underlying issues with emotional suppression and limited cognitive resources to improve risk perception abilities. Limitations and future directions are discussed.

Analysis of expressed sequence tags from the agarophyte Gracilaria tenuistipitata (Rhodophyta)

A total of 3,631 expressed sequence tags (ESTs) were established from two size-selected cDNA libraries made from the tetrasporophytic phase of the agarophytic red alga Gracilaria tenuistipitata. The average sizes of the inserts in the two libraries were 1,600 bp and 600 bp, with an average length of the edited sequences of 850 bp. Clustering gave 2,387 assembled sequences with a redundancy of 53%. Of the ESTs, 65% had significant matches to sequences deposited in public databases, 11% to proteins without known function, and 35% were novel. The most represented ESTs were a Na/K-transporting ATPase, a hedgehog-like protein, a glycine dehydrogenase and an actin. Most of the identified genes were involved in primary metabolism and housekeeping. The largest functional group was thus genes involved in metabolism with 14% of the ESTs; other large functional categories included energy, transcription, and protein synthesis and destination. The codon usage was examined using a subset of the data, and the codon bias was found to be limited with all codon combinations used.

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

The pattern recognition receptors expressed on neutrophils and the associated cytokine profile from different aged patients with Candida-related denture stomatitis

This study evaluated the expression of pattern recognition receptors (PRRs) and activation factors associated with salivary and blood neutrophils from different aged patients diagnosed with Candida-related denture stomatitis (DS). Expression of neutrophil PRRs was determined by flow cytometry and immunofluorescence, and the levels of selected cytokines that influence immune activation were determined by ELISA. The salivary (but not the serum derived) neutrophils of individuals with DS were found to have an increased expression of CD69 regardless of the age of the patient compared to patients without DS. However, these salivary neutrophils had a lower expression of CD66b and CD64. Expression of TLR2 was lower on the salivary-and serum-derived neutrophils from elderly individuals compared to the neutrophils of younger subjects, regardless of whether the individual had DS. Salivary interleukin (IL)-4 was elevated in both of the elderly subject groups (with or without DS). Only elderly DS patients were observed to have increased serum IL-4 levels and reduced salivary IL-12 levels. Younger DS patients showed an increase in salivary IL-10 levels, and both the saliva and the serum levels of IFN-gamma were increased in all of the younger subjects. Our data demonstrated that changes in both the oral immune cells and the protein components could be associated with DS. Furthermore, changes in the blood-derived factors were more associated with age than DS status. (C) 2012 Elsevier Inc. All rights reserved.

COMT Met (158) modulates facial emotion recognition in bipolar I disorder mood episodes

Background: One of the many cognitive deficits reported in bipolar disorder (BD) patients is facial emotion recognition (FER), which has recently been associated with dopaminergic catabolism. Catechol-O-methyltransferase (COMT) is one of the main enzymes involved in the metabolic degradation of dopamine (DA) in the prefrontal cortex (PFC). The COMT gene polymorphism rs4680 (Val(158)Met) Met allele is associated with decreased activity of this enzyme in healthy controls. The objective of this study was to evaluate the influence of Val(158)Met on FER during manic and depressive episodes in BD patients and in healthy controls. Materials and methods: 64 BD type I patients (39 in manic and 25 in depressive episodes) and 75 healthy controls were genotyped for COMT rs4680 and assessed for FER using the Ekman 60 Faces (EK60) and Emotion Hexagon (Hx) tests. Results: Bipolar manic patients carrying the Met allele recognized fewer surprised faces, while depressed patients with the Met allele recognized fewer "angry" and "happy" faces. Healthy homozygous subjects with the Met allele had higher FER scores on the Hx total score, as well as on "disgust" and "angry" faces than other genotypes. Conclusion: This is the first study suggesting that COMT rs4680 modulates FER differently during BD episodes and in healthy controls. This provides evidence that PFC DA is part of the neurobiological mechanisms of social cognition. Further studies on other COMT polymorphisms that include euthymic BD patients are warranted. ClinicalTrials.gov Identifier: NCT00969. (C) 2011 Elsevier B.V. All rights reserved.

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

Abstract Background Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. Results In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-β-glucosidase) in mycelium cells; and ags (an α-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. Conclusion Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.