Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the sigma(54) regulon

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a sigma(54)-dependent manner. A more complete picture of the sigma(54) regulon was achieved by combining the transcriptome data with an in silico search for potential sigma(54)-dependent promoters, using a position weight matrix approach. One of these sigma(54)-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a sigma(54)-dependent promoter. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the sigma(54) regulon.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Characterization of new IS elements and studies of their dispersion in two subspecies of Leifsonia xyli

Background: Leifsonia xyli is a xylem-inhabiting bacterial species comprised of two subspecies: L. xyli subsp. xyli (Lxx) and L. xyli subsp. cynodontis (Lxc). Lxx is the causal agent of ratoon stunting disease in sugarcane commercial fields and Lxc colonizes the xylem of several grasses causing either mild or no symptoms of disease. The completely sequenced genome of Lxx provided insights into its biology and pathogenicity. Since IS elements are largely reported as an important source of bacterial genome diversification and nothing is known about their role in chromosome architecture of L. xyli, a comparative analysis of Lxc and Lxx elements was performed. Results: Sample sequencing of Lxc genome and comparative analysis with Lxx complete DNA sequence revealed a variable number of IS transposable elements acting upon genomic diversity. A detailed characterization of Lxc IS elements and a comparative review with IS elements of Lxx are presented. Each genome showed a unique set of elements although related to same IS families when considering features such as similarity among transposases, inverted and direct repeats, and element size. Most of the Lxc and Lxx IS families assigned were reported to maintain transposition at low levels using translation regulatory mechanisms, consistent with our in silico analysis. Some of the IS elements were found associated with rearrangements and specific regions of each genome. Differences were also found in the effect of IS elements upon insertion, although none of the elements were preferentially associated with gene disruption. A survey of transposases among genomes of Actinobacteria showed no correlation between phylogenetic relatedness and distribution of IS families. By using Southern hybridization, we suggested that diversification of Lxc isolates is also mediated by insertion sequences in probably recent events. Conclusion: Collectively our data indicate that transposable elements are involved in genome diversification of Lxc and Lxx. The IS elements were probably acquired after the divergence of the two subspecies and are associated with genome organization and gene contents. In addition to enhancing understanding of IS element dynamics in general, these data will contribute to our ongoing comparative analyses aimed at understanding the biological differences of the Lxc and Lxx.

HFE gene mutations in patients with primary iron overload: Is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation >50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and beta 2-microglobulin ((beta 2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations. (C) 2010 Elsevier Inc. All rights reserved.

Intrinsically bent DNA sites in the Drosophila melanogaster third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-beta. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-beta region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes.

Evaluation of RET polymorphisms in a six-generation family with G533C RET mutation: specific RET variants may modulate age at onset and clinical presentation

P>Context We previously described a six-generation family with G533C RET mutation and medullary thyroid carcinoma, in the largest family reported do date. Of particular interest, phenotype variability regarding the age of onset and clinical presentation of the disease, was observed. Objective We evaluate whether single SNPs within RET oncogene or haplotype comprising the RET variants (defined by Haploview) could predispose to early development of MTC in this family and influence the clinical manifestation. Design Eight SNPs were selected based on their previous association with the clinical course of hereditary or sporadic MTC, in particular promoting an early onset of disease. The variants were initially tested in 77 G533C-carriers and 100 controls using either PCR-direct sequencing or PCR-RFLP. Association between a SNP or haplotype and age at diagnosis or presence of lymph node metastasis was tested in 34 G533C-carries with MTC. Different bioinformatic tools were used to evaluate the potential effects on RNA splicing. Results An association was found between IVS1-126G > T and age at diagnosis. The variant [IVS8 +82A > G; 85-86 insC] was associated with the presence of lymph node metastases at diagnosis. In silico analysis suggested that this variant may induce abnormal splicing. This in silico analysis predicted that the [IVS8 +82A > G; 85-86 insC] could alter the splicing by disrupting and/or creating exonic splicing enhancer motifs. Conclusions We here identified two RET variants that were associated with phenotype variability in G533C-carriers, which highlights the fact that the modifier effect of a variant might depend on the type of mutation.

Axon navigation in the mammalian primary olfactory pathway: Where to next?

The process of establishing long-range neuronal connections can be divided into at least three discrete steps. First, axons need to be stimulated to grow and this growth must be towards appropriate targets. Second, after arriving at their target, axons need to be directed to their topographically appropriate position and in some cases, such as in cortical structures, they must grow radially to reach the correct laminar layer Third, axons then arborize and form synaptic connections with only a defined subpopulation of potential post-synaptic partners. Attempts to understand these mechanisms in the visual system have been ongoing since pioneer studies in the 1940s highlighted the specificity of neuronal connections in the retino-tectal pathway. These classical systems-based approaches culminated in the 1990s with the discovery that Eph-ephrin repulsive interactions were involved in topographical mapping. In marked contrast, it was the cloning of the odorant receptor family that quickly led to a better understanding of axon targeting in the olfactory system. The last 10 years have seen the olfactory pathway rise in prominence as a model system for axon guidance. Once considered to be experimentally intractable, it is now providing a wealth of information on all aspects of axon guidance and targeting with implications not only for our understanding of these mechanisms in the olfactory system but also in other regions of the nervous system.

Cultivo de Eucalyptus urograndis em atmosfera enriquecida com CO2: mudanças no proteoma cloroplastidial

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

Novel FGFR1 Mutations in Kallmann Syndrome and Normosmic Idiopathic Hypogonadotropic Hypogonadism: Evidence for the Involvement of an Alternatively Spliced Isoform

OBJECTIVE: To determine the prevalence of fibroblast growth factor receptor 1 (FGFR1) mutations and their predicted functional consequences in patients with idiopathic hypogonadotropic hypogonadism (IHH). DESIGN: Cross-sectional study. SETTING: Multicentric. PATIENT(S): Fifty unrelated patients with IHH (21 with Kallmann syndrome and 29 with normosmic IHH). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Patients were screened for mutations in FGFR1. The functional consequences of mutations were predicted by in silico structural and conservation analysis. RESULT(S): Heterozygous FGFR1 mutations were identified in six (12%) kindreds. These consisted of frameshift mutations (p.Pro33-Alafs*17 and p.Tyr654*) and missense mutations in the signal peptide (p.Trp4Cys), in the D1 extracellular domain (p.Ser96Cys) and in the cytoplasmic tyrosine kinase domain (p.Met719Val). A missense mutation was identified in the alternatively spliced exon 8A (p.Ala353Thr) that exclusively affects the D3 extracellular domain of FGFR1 isoform IIIb. Structure-based and sequence-based prediction methods and the absence of these variants in 200 normal controls were all consistent with a critical role for the mutations in the activity of the receptor. Oligogenic inheritance (FGFR1/CHD7/PROKR2) was found in one patient. CONCLUSION(S): Two FGFR1 isoforms, IIIb and IIIc, result from alternative splicing of exons 8A and 8B, respectively. Loss-of-function of isoform IIIc is a cause of IHH, whereas isoform IIIb is thought to be redundant. Ours is the first report of normosmic IHH associated with a mutation in the alternatively spliced exon 8A and suggests that this disorder can be caused by defects in either of the two alternatively spliced FGFR1 isoforms.

On and off air: an ethnographic exploration of minority radio in Portugal

Taking a Media Anthropology’s approach to dynamics of mediated selfrepresentation in migratory contexts, this thesis starts by mapping radio initiatives produced by, for and/or with migrants in Portugal. To further explore dynamics of support of initial settlement in the country, community-making, cultural reproduction, and transnational connectivity - found both in the mapping stage and the minority media literature (e.g. Kosnick, 2007; Rigoni & Saitta, 2012; Silverstone & Georgiou, 2005) - a case study was selected: the station awarded with the first bilingual license in Portugal. The station in question caters largely to the British population presenting themselves as “expats” and residing in the Algarve. The ethnographic strategy to research it consisted of “following the radio” (Marcus, 1995) beyond the station and into the events and establishments it announces on air, so as to relate production and consumption realms. The leading research question asks how does locally produced radio play into “expats” processes of management of cultural identity – and what are the specificities of its role? Drawing on conceptualizations of lifestyle migration (Benson & O’Reilly, 2009), production of locality (Appadurai 1996) and the public sphere (Butsch, 2007; Calhoun & et al, 1992; Dahlgren, 2006), this thesis contributes to valuing radio as a productive gateway to research migrants’ construction of belonging, to inscribe a counterpoint in the field of minority media, and to debate conceptualizations of migratory categories and flows. Specifically, this thesis argues that the station fulfills similar roles to other minority radio initiatives but in ways that are specific to the population being catered to. Namely, unlike other minority stations, radio facilitates the process of transitioning between categories along on a continuum linking tourists and migrants. It also reflects and participates in strategies of reterritorialization that rest on functional and partial modes of incorporation. While contributing to sustain a translocality (Appadurai, 1996) it indexes and fosters a stance of connection that is symbolically and materially connected to the UK and other “neighborhoods” but is, simultaneously, oriented to engaging with the Algarve as “home”. Yet, besides reifying a British cultural identity, radio’s oral, repetitive and ephemeral discourse particularly trivializes the reproduction of an ambivalent stance of connection with place that is shared by other “expats”. This dynamic is related to migratory projects driven by social imaginaries fostered by international media that stimulate the search for idealized ways of living, which the radio associates with the Algarve. While recurrently localizing and validating the narrative projecting an idealized “good life”, radio amplifies dynamics among migrants that seem to reaffirm the migratory move as a good choice.

RNA-seq analysis of the Quercus suber root response to drought

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepreneurship

Understanding adaptation to pH in an industrially relevant glycoside hydrolase family 8 xylanase

Dissertação de mestrado em Genética Molecular

Useful clinical features for the selection of ideal patients with strial fibrillation for mapping and catheter ablation

OBJECTIVE: To identify useful clinical characteristics for selecting patients eligible for mapping and ablation of atrial fibrillation. METHODS: We studied 9 patients with atrial fibrillation, without structural heart disease, associated with: 1) antiarrhythmic drugs, 2) symptoms of low cardiac output, and 3) intention to treat. Seven patients had paroxysmal atrial fibrillation and 2 had recurrent atrial fibrillation. RESULTS: In the 6 patients who underwent mapping (all had paroxysmal atrial fibrillation), catheter ablation was successfully carried out in superior pulmonary veins in 5 patients (the first 3 in the left superior pulmonary vein and the last 2 in the right superior pulmonary vein). One patient experienced a recurrence of atrial fibrillation after 10 days. We observed that patients who had short episodes of atrial fibrillation on 24-hour Holter monitoring before the procedure were those in whom mapping the focus of tachycardia was possible. Tachycardia was successfully suppressed in 4 of 6 patients. The cause of failure was due to the impossibility of maintaining sinus rhythm long enough for efficient mapping. CONCLUSION: Patients experiencing short episodes of atrial fibrillation during 24-hour Holter monitoring were the most eligible for mapping and ablation, with a final success rate of 66%, versus the global success rate of 44%. Patients with persistent atrial fibrillation were not good candidates for focal ablation.

Mutation screening of the glutamate cysteine ligase modifier (GCLM) gene in patients with schizophrenia.

BACKGROUND: Experimental evidences show that glutathione and its rate-limiting synthesizing enzyme, the glutamate-cysteine ligase (GCL), are involved in the pathogenesis of schizophrenia. Furthermore, genetic association has been previously reported between two single nucleotide polymorphisms lying in noncoding regions of glutamate cysteine ligase modifier (GCLM) gene, which specifies for the modifier subunit of GCL and schizophrenia. OBJECTIVE: We wanted to investigate the presence of GCLM true functional mutations, likely in linkage disequilibrium with the previously identified single nucleotide polymorphism alleles, in the same set of cases that allowed the detection of the original association signal. METHODS: We screened all the coding regions of GCLM and their intronic flanking vicinities in 353 patients with schizophrenia by direct DNA sequencing. RESULTS: Ten sequence variations were identified, five of which were not previously described. None of these DNA changes was within the GCLM coding sequence and in-silico analysis failed to indicate functional impairment induced by these variations. Furthermore, screening of normal controls and downstream statistical analyses revealed no significant relationship of any of these DNA variants with schizophrenia. CONCLUSION: It is unlikely that functional mutations in the GCLM gene could play a major role in genetic predisposition to schizophrenia and further studies will be required to assess its etiological function in the disease.