Estudos de expressão gênica utilizando-se microarrays: delineamento, análise, e aplicações na pesquisa zootécnica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Optimal clone identifier for genomic shotgun libraries: OC Identifier tool

In DNA microarray experiments, the gene fragments that are spotted on the slides are usually obtained by the synthesis of specific oligonucleotides that are able to amplify genes through PCR. Shotgun library sequences are an alternative to synthesis of primers for the study of each gene in the genome. The possibility of putting thousands of gene sequences into a single slide allows the use of shotgun clones in order to proceed with microarray analysis without a completely sequenced genome. We developed an OC Identifier tool (optimal clone identifier for genomic shotgun libraries) for the identification of unique genes in shotgun libraries based on a partially sequenced genome; this allows simultaneous use of clones in projects such as transcriptome and phylogeny studies, using comparative genomic hybridization and genome assembly. The OC Identifier tool allows comparative genome analysis, biological databases, query language in relational databases, and provides bioinformatics tools to identify clones that contain unique genes as alternatives to primer synthesis. The OC Identifier allows analysis of clones during the sequencing phase, making it possible to select genes of interest for construction of a DNA microarray. ©FUNPEC-RP.

Effects of BST and high energy diet on gene expression in mammary parenchyma of dairy heifers

The objective of this study was to determine the effects of dietary energy and recombinant bovine somatotropin (bST) injection to identify genes that might control mammogenesis. Total RNA was extracted from the parenchymal tissue of 32 heifers randomly assigned to one of four treatments: two diets (a standard diet and a high energy, high protein diet), each with or without bST. To perform microarray experiments, RNA samples were pooled (2 animals/pool) before reverse transcription and labeling with Cy3 or Cy5. A 4-node loop design was used to examine the differential gene expression among treatments using a bovine-specific cDNA micro array (National Bovine Functional Genomics Consortium Library, NBFGC) containing 18,263 unique expressed sequence tags (EST). Significance levels of differential gene expression among treatments were assessed using a mixed model approach. Injection of bST altered the expression of 12 % of the genes on NBFGC slide related to tissue development, whereas 6% were altered by diet. Administration of bST increases the expression of genes positively related to cell proliferation and mammary parenchyma to a greater extent than a high energy diet. © 2013 Sociedade Brasileira de Zootecnia.

Aplicação de modelos lineares para análise de expressão gênica em experimentos de microarrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

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

Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides

The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1.2 mm apart. Section of 4 mu m were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer's recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (>80%), almost complete (75-79%), or partial (50-74%). The commercial adhesive tape group presented lesser total loss (1.1 versus 6.4%), almost complete loss (2.2 versus 3.5%), and partial loss (2.1 versus 3.8%) than the silanized slide group (ANOVA, P < 0.05). The sum of total and almost complete losses in the silanized slide group was 9.9%, greater than the losses in slides using commercial adhesive tapes (3.3%) and less than reported and considered acceptable in the literature (10-30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research.

Some experiments with high order compact methods using a computer algebra software - Part I

We consider a procedure for obtaining a compact fourth order method to the steady 2D Navier-Stokes equations in the streamfunction formulation using the computer algebra system Maple. The resulting code is short and from it we obtain the Fortran program for the method. To test the procedure we have solved many cavity-type problems which include one with an analytical solution and the results are compared with results obtained by second order central differences to moderate Reynolds numbers. (c) 2005 Elsevier B.V. All rights reserved.

Some experiments with high order compact methods using a computer algebra software - Part II (non-uniform grid)

We generalize a procedure proposed by Mancera and Hunt [P.F.A. Mancera, R. Hunt, Some experiments with high order compact methods using a computer algebra software-Part 1, Appl. Math. Comput., in press, doi: 10.1016/j.amc.2005.05.015] for obtaining a compact fourth-order method to the steady 2D Navier-Stokes equations in the streamfunction formulation-vorticity using the computer algebra system Maple, which includes conformal mappings and non-uniform grids. To analyse the procedure we have solved a constricted stepped channel problem, where a fine grid is placed near the re-entrant corner by transformation of the independent variables. (c) 2006 Elsevier B.V. All rights reserved.

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12 000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.

Stochastic dynamics in exotic and native blowflies: an analysis combining laboratory experiments and a two-patch metapopulation model

In this study we explored the stochastic population dynamics of three exotic blowfly species, Chrysomya albiceps, Chrysomya megacephala and Chrysomya putoria, and two native species, Cochliomyia macellaria and Lucilia eximia, by combining a density-dependent growth model with a two-patch metapopulation model. Stochastic fecundity, survival and migration were investigated by permitting random variations between predetermined demographic boundary values based on experimental data. Lucilia eximia and Chrysomya albiceps were the species most susceptible to the risk of local extinction. Cochliomyia macellaria, C. megacephala and C. putoria exhibited lower risks of extinction when compared to the other species. The simultaneous analysis of stochastic fecundity and survival revealed an increase in the extinction risk for all species. When stochastic fecundity, survival and migration were simulated together, the coupled populations were synchronized in the five species. These results are discussed, emphasizing biological invasion and interspecific interaction dynamics.

Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila

Insecticide resistance in laboratory selected Drosophila strains has been associated with upregulation of a range of different cytochrome P450s, however in recent field isolates of D. melanogaster resistance to DDT and other compounds is conferred by one P450 gene, Cyp6g1. Using microarray analysis of all Drosophila P450 genes, here we show that different P450 genes such as Cyp12d1 and Cyp6a8 can also be selected using DDT in the laboratory. We also show, however, that a homolog of Cyp6g1 is over-expressed in a field resistant strain of D. simulans. In order to determine why Cyp6g1 is so widely selected in the field we examine the pattern of cross-resistance of both resistant strains and transgenic flies over-expressing Cyp6g1 alone. We show that all three DDT selected P450s can confer resistance to the neonicotinoid imidacloprid but that Cyp6a8 confers no cross-resistance to malathion. Transgenic flies over-expressing Cyp6g1 also show cross-resistance to other neonicotinoids such as acetamiprid and nitenpyram. We suggest that the broad level of cross-resistance shown by Cyp6g1 may have facilitated its selection as a resistance gene in natural Drosophila populations. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Complementarity of eastern and western hemisphere long-baseline neutrino oscillation experiments

We present a general formalism for extracting information on the fundamental parameters associated with neutrino masses and mixings from two or more long baseline neutrino oscillation experiments. This formalism is then applied to the current most likely experiments using neutrino beams from the Japan Hadron Facility (JHF) and Fermilab's NuMI beamline. Different combinations of muon neutrino or muon anti-neutrino running are considered. The type of neutrino mass hierarchy is extracted using the effects of matter on neutrino propogation. Contrary to naive expectation, we find that both beams using neutrinos is more suitable for determining the hierarchy provided that the neutrino energy divided by baseline (E/L) for NuMI is smaller than or equal to that of JHF, whereas to determine the small mixing angle, theta(13), and the CP or T violating phase delta, one neutrino and the other anti-neutrino are most suitable. We make extensive use of bi-probability diagrams for both understanding and extracting the physics involved in such comparisons.

Method for determination of vertical bar U(e)3 vertical bar in neutrino oscillation appearance experiments

We point out that determination of the MNS matrix element \U-e3\ = s(13) in long-baseline nu(mu) --> nu(e) neutrino oscillation experiments suffers from large intrinsic uncertainty due to the unknown CP violating phase delta and sign of Deltam(13)(2). We propose a new strategy for accurate determination of theta(13); tune the beam energy at the oscillation maximum and do the measurement both in neutrino and antineutrino channels. We show that it automatically resolves the problem of parameter ambiguities which involves delta, theta(13), and the sign of Deltam(13)(2). (C) 2002 Elsevier B.V. B.V. All rights reserved.