Biomedical subjects

Guilherme J M Rosa

Publications and source records attributed to Guilherme J M Rosa.

10 recordsLinked to original sources

PubMed2006

Review of microarray experimental design strategies for genetical genomics studies.

Genetical genomics approaches provide a powerful tool for studying the genetic mechanisms governing variation in complex traits. By combining information on phenotypic traits, pedigree structure, molecular markers, and gene expression, such studies can be used for estimating heritability of mRNA transcript abundances, for mapping expression quantitative trait loci (eQTL), and for inferring regulatory gene networks. Microarray experiments, however, can be extremely costly and time consuming, which may limit sample sizes and statistical power. Thus it is crucial to optimize experimental designs by carefully choosing the subjects to be assayed, within a selective profiling approach, and by cautiously controlling systematic factors affecting the system. Also, a rigorous strategy should be used for allocating mRNA samples across assay batches, slides, and dye labeling, so that effects of interest are not confounded with nuisance factors. In this presentation, we review some selective profiling strategies for genetical genomics studies, including the selection of individuals for increased genetic dissimilarity and for a higher number of recombination events. Efficient designs for studying epistasis are also discussed, as well as experiments for inferring heritability of transcriptional levels. It is shown that solving an optimal design problem generally requires a numerical implementation and that the optimality criteria should be intimately related to the goals of the experiment, such as the estimation of additive, dominance, and interacting effects, localizing putative eQTL, or inferring genetic and environmental variance components associated with transcriptional abundances.

Animals↗

PubMed2006

The transcriptome of human oocytes.

The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis.

Animals↗

PubMed2006

Design of microarray experiments for genetical genomics studies.

Microarray experiments have been used recently in genetical genomics studies, as an additional tool to understand the genetic mechanisms governing variation in complex traits, such as for estimating heritabilities of mRNA transcript abundances, for mapping expression quantitative trait loci, and for inferring regulatory networks controlling gene expression. Several articles on the design of microarray experiments discuss situations in which treatment effects are assumed fixed and without any structure. In the case of two-color microarray platforms, several authors have studied reference and circular designs. Here, we discuss the optimal design of microarray experiments whose goals refer to specific genetic questions. Some examples are used to illustrate the choice of a design for comparing fixed, structured treatments, such as genotypic groups. Experiments targeting single genes or chromosomic regions (such as with transgene research) or multiple epistatic loci (such as within a selective phenotyping context) are discussed. In addition, microarray experiments in which treatments refer to families or to subjects (within family structures or complex pedigrees) are presented. In these cases treatments are more appropriately considered to be random effects, with specific covariance structures, in which the genetic goals relate to the estimation of genetic variances and the heritability of transcriptional abundances.

Animals↗

PubMed2006

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD).

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (approximately 17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course (P<0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold (P<0.01) revealed 35 genes (approximately 3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-gamma gene (IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated PBMC provides the first description of an M. bovis-specific signature of infection that may provide insights into the molecular basis of the host response to infection. Although the present study was carried out with mixed PBMC cell populations, it will guide future studies to dissect immune cell-specific gene expression patterns in response to M. bovis infection.

Animals↗

PubMed2006

Glucocorticoid modulation of Bcl-2 family members A1 and Bak during delayed spontaneous apoptosis of bovine blood neutrophils.

Neutrophils are critical for innate immune defense against microbial invasion but can also cause inflammatory tissue damage if their life span is not tightly regulated. Antiinflammatory glucocorticoids delay spontaneous apoptosis in human, rodent, and bovine neutrophils, but mechanisms involved are unknown. We hypothesized here that glucocorticoids delay neutrophil apoptosis by altering expression of key Bcl-2 apoptosis regulatory proteins, A1 and Bak, via activation of the cell's glucocorticoid receptors. To test this hypothesis, isolated bovine blood neutrophils were exposed to dexamethasone with and without glucocorticoid receptor antagonism (RU486) and aged ex vivo over 0-24 h for assessment of various spontaneous apoptosis pathway indicators and A1 and Bak abundance. Results show that dexamethasone preserved neutrophil mitochondrial membrane integrity, delayed caspase-9 activation, and reduced the rate of spontaneous apoptosis. Also, dexamethasone increased A1 and decreased Bak mRNA abundance. RU486 pretreatment of the cells abrogated each of these dexamethasone effects. Dexamethasone-induced increases in A1 mRNA were reflected in A1 protein increases, which also were observed in circulating neutrophils of dexamethasone-treated animals. Bak protein decreases were observed in neutrophils of the dexamethasone-treated animals but not in isolated neutrophils, suggesting that stimuli additional to (and perhaps regulated by) glucocorticoid are required to affect Bak protein expression changes in neutrophils. Collectively, our results are unique in demonstrating a mechanism behind glucocorticoid regulation of spontaneous apoptosis and implicate steroid receptor activation and subsequent regulation of A1 and Bak as contributors to mitochondrial membrane stability, reduced caspase-9 activity, and delayed apoptosis in bovine neutrophils exposed to glucocorticoids.

Animals↗

PubMed2006

Transcriptome of local innate and adaptive immunity during early phase of infectious bronchitis viral infection.

To understand the mechanistic basis of local innate and adaptive immunity against infectious bronchitis virus (IBV) at the molecular level, we examined the gene transcription profile of tracheal epithelial layers 3 d after infection of chickens with an attenuated IBV-Massachusetts strain. Results suggested that the transcription levels of 365 genes were either upregulated or downregulated (2-fold and higher) after IBV infection. Among the upregulated 250 genes, 25 were directly immune-related genes. These upregulated immune response genes included TLR2, TLR3, interferon-induced antiviral genes (Mx), and genes responsible for cytotoxic T cell killing such as Fas antigen and granzyme-A. Overall, a diversity of innate immunity and helper T cell type 1 (Th1)-biased adaptive immunity are activated in the host's early defense against IBV invasion, and they are responsible for the rapid clearance of virus from the local infection.

Animals↗

PubMed2005

On reference designs for microarray experiments.

We compare four variants of the reference design for microarray experiments in terms of their relative efficiency. A common reference sample across arrays is the most extensively used variation in practice, but independent samples from a reference group have also been considered in previous works. The relative efficiency of these designs depends of the number of treatments and the ratio between biological and technical variances. Here, we propose another alternative of reference structure, denoted by blocked reference design (BRD), in which each set (replication) of the treated samples is co-hybridized to an independent experimental unit of the control (reference) group. We provide efficiency curves for each pair of designs under different scenarios of variance ratio and number of treatments groups. The results show that the BRD is more efficient and less expensive than the traditional reference designs. Among the situations where the BRD is likely to be preferable we list time course experiments with a baseline and drug experiments with a placebo group.

Journal Article↗

PubMed2004

Microarray analysis of gene expression in blood neutrophils of parturient cows.

It is well documented that blood neutrophils from parturient dairy cows do not perform as well as neutrophils from nonparturient cows in laboratory assays of adhesion, migration, or phagocytosis-induced respiratory burst. However, little is known about the possible molecular basis for parturition-induced changes in neutrophils. cDNA microarray analysis was used in the current study to explore parturition-induced changes in gene expression profiles in bovine blood neutrophils. Total RNA from isolated blood neutrophils of four parturient Holstein cows was obtained before, during, and after parturition, reverse transcribed into cDNA, and sequentially labeled with Cy3 or Cy5 dyes prior to paired hybridizations to 1,056 member bovine total leukocyte (BOTL-3) microarrays in a loop design. Resulting gene expression data were LOWESS normalized by array and analyzed using a mixed model approach. Results showed that expression profiles for 302 BOTL-3 genes were influenced by parturition. BLASTn analysis and preliminary clustering of affected genes by biological function indicated that the largest proportion (14%) of changed genes encode proteins critical to regulation of apoptosis. Independent confirmation of altered expression for 16 of these genes was achieved using quantitative real-time RT-PCR (Q-RT-PCR). A predominantly survival phenotype inferred from the microarray and Q-RT-PCR results was substantiated by monitoring apoptosis status of blood neutrophils from castrated male cattle cultured in the presence of sera from parturient cows. Thus our combined gene expression and apoptosis phenotyping results suggest that bovine parturition may induce prolonged survival in normally short-lived blood neutrophils.

Animals↗

PubMed2003

Evidence for a novel gene expression program in peripheral blood mononuclear cells from Mycobacterium avium subsp. paratuberculosis-infected cattle.

A bovine-specific cDNA microarray system was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from control uninfected (n = 4) and Johne's disease-positive (n = 6) Holstein cows. Microarray experiments were designed so that for each animal, a direct comparison was made between PBMCs stimulated in vitro with Mycobacterium avium subsp. paratuberculosis and PBMCs stimulated with phosphate-buffered saline (nil-stimulated PBMCs). As expected, M. avium subsp. paratuberculosis stimulation of infected cow PBMCs enhanced expression of gamma interferon transcripts. In addition, expression of 15 other genes was significantly affected (>1.25-fold change; P < 0.05) by in vitro stimulation with M. avium subsp. paratuberculosis. Similar treatment of control cow PBMCs with M. avium subsp. paratuberculosis resulted in significant changes in expression of 13 genes, only 2 of which were also affected in PBMCs from the infected cow PBMCs. To compare gene expression patterns in the two cow infection groups (infected cows and uninfected cows), a mixed-model analysis was performed with the microarray data. This analysis indicated that there were major differences in the gene expression patterns between cells isolated from the two groups of cows, regardless of in vitro stimulation. A total of 86 genes were significantly differentially expressed (P < 0.01) in M. avium subsp. paratuberculosis-stimulated PBMCs from infected cows compared to expression in similarly treated PBMCs from control cows. Surprisingly, a larger number of genes (110 genes) were also found to be significantly differentially expressed (P < 0.01) in nil-stimulated cells from the two infection groups. The expression patterns of selected genes were substantiated by quantitative real-time reverse transcriptase PCR. Flow cytometric analysis indicated that there were no gross differences in the relative populations of major immune cell types in PBMCs from infected and control cows. Thus, data presented in this report indicate that the gene expression program of PBMCs from M. avium subsp. paratuberculosis-infected cows is inherently different from that of cells from control uninfected cows.

Animals↗

PubMedDate not supplied

A Bayesian approach for constructing genetic maps when markers are miscoded.

The advent of molecular markers has created opportunities for a better understanding of quantitative inheritance and for developing novel strategies for genetic improvement of agricultural species, using information on quantitative trait loci (QTL). A QTL analysis relies on accurate genetic marker maps. At present, most statistical methods used for map construction ignore the fact that molecular data may be read with error. Often, however, there is ambiguity about some marker genotypes. A Bayesian MCMC approach for inferences about a genetic marker map when random miscoding of genotypes occurs is presented, and simulated and real data sets are analyzed. The results suggest that unless there is strong reason to believe that genotypes are ascertained without error, the proposed approach provides more reliable inference on the genetic map.

Bayes Theorem↗