Luciane Madureira de Almeida

Microarray analysis of differentially expressed genes from Peyer's patches of cattle orally challenged with bovine spongiform encephalopathy.

The most likely route of entry of infection following oral exposure to transmissible spongiform encephalopathies (TSE) is via the immunologically active Peyers patches (PP). These secondary lymphoid organs appear to be the potential route for prion neuroinvasion. However, the molecular mechanisms involved in the uptake of the infectious prion agent and progression of disease remain still unclear. This investigation examined the changes in gene expression in PP following oral exposure of cattle to bovine spongiform encephalopathy (BSE) agents. The gene expression patterns in PP from cows 12 mo after BSE challenge were compared with controls using a microarray platform containing 24,000 oligonucleotides representing 16,846 unique gene loci and 5943 Expressed Sequence Tag (EST) from bovine genome. Between the challanged and control animals, 90 genes and 16 EST were identified as significantly differentially, expressed (>2.0-fold change): 36 were upregulated and 70 were downregulated. Of these genes, five were found to be related to immune function. Major histocompatibility complex (MHC) class II, MHC class II DQ alpha, L-RAP, and two hypothetical proteins. Differentially expressed genes related to cellular and metabolic processes including development and maturation of cells in the PP were also identified. In this context, the potential impacts of these gene expression changes in PP on BSE development are discussed.

Gene Expression in the Medulla Following Oral Infection of Cattle with Bovine Spongiform Encephalopathy

The identification of variations in gene expression in response to bovine spongiform encephalopathy (BSE) may help to elucidate the mechanisms of neuropathology and prion replication and discover biomarkers for disease. In this study, genes that are differentially expressed in the caudal medulla tissues of animals infected with different doses of PrPBSE at 12 and 45 mo post infection were compared using array containing 24,000 oligonucleotide probes. Data analysis identified 966 differentially expressed (DE) genes between control and infected animals. Genes identified in at least two of four experiments (control versus 1-g infected animals at 12 and 45-mo; control versus 100-g infected animals at 12 and 45 mo) were considered to be the genes that may be associated with BSE disease. From the 176 DE genes associated with BSE, 84 had functions described in the Gene Ontology (GO) database. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 14 genes revealed that prion infection may cause dysfunction of several different networks, including extracellular matrix (ECM), cell adhesion, neuroactive ligand–receptor interaction, complement and coagulation cascades, MAPK signaling, neurodegenerative disorder, SNARE interactions in vesicular transport, and the transforming growth factor (TGF) beta signaling pathways. The identification of DE genes will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species. Additional studies on larger number of animals are in progress in our laboratory to investigate the roles of these DE genes in pathogenesis of BSE.

Microarray analysis in caudal medulla of cattle orally challenged with bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is a fatal disorder in cattle characterized by progressive neurodegeneration of the central nervous system. We investigated the molecular mechanisms involved in neurodegeneration during prion infection through the identification of genes that are differentially expressed (DE) between experimentally infected and non-challenged cattle. Gene expression of caudal medulla from control and orally infected animals was compared by microarray analysis using 24,000 bovine oligonucleotides representing 16,846 different genes to identify DE genes associated with BSE disease. In total, 182 DE genes were identified between normal and BSE-infected tissues (>2.0-fold change, P < 0.01); 81 DE genes had gene ontology functions, which included synapse function, calcium ion regulation, immune and inflammatory response, apoptosis, and cytoskeleton organization; 13 of these genes were found to be involved in 26 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression of five DE genes associated with synapse function (tachykinin, synuclein, neuropeptide Y, cocaine, amphetamine-responsive transcript, and synaptosomal-associated protein 25 kDa) and three DE genes associated with calcium ion regulation (parvalbumin, visinin-like, and cadherin) was further validated in the medulla tissue of cattle at different infection times (6, 12, 42, and 45 months post-infection) by qRT-PCR. These data will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species.

Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes upregulated and 197 genes downregulated. There were 249 DE genes in the spleen (168 up- and 81 downregulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 downregulated), and 55 DE genes in the tonsil (21 up- and 34 downregulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.

Transcriptome analysis of the medulla tissue from cattle in response to bovine spongiform encephalopathy using digital gene expression tag profiling

Bovine spongiform encephalopathy (BSE) is a transmissible, fatal neurodegenerative disorder of cattle produced by prions. The use of excessive parallel sequencing for comparison of gene expression in bovine control and infected tissues may help to elucidate the molecular mechanisms associated with this disease. In this study, tag profiling Solexa sequencing was used for transcriptome analysis of bovine brain tissues. Replicate libraries were prepared from mRNA isolated from control and infected (challenged with 100 g of BSE-infected brain) medulla tissues 45 mo after infection. For each library, 5–6 million sequence reads were generated and approximately 67–70% of the reads were mapped against the Bovine Genome database to approximately 13,700–14,120 transcripts (each having at least one read). About 42–47% of the total reads mapped uniquely. Using the GeneSifter software package, 190 differentially expressed (DE) genes were identified (>2.0-fold change, p < .01): 73 upregulated and 117 downregulated. Seventy-nine DE genes had functions described in the Gene Ontology (GO) database and 16 DE genes were involved in 38 different pathways described in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Digital analysis expression by tag profiling may be a powerful approach to comprehensive transcriptome analysis to identify changes associated with disease progression, leading to a better understanding of the underlying mechanism of pathogenesis of BSE.

Comparative study of bone tissue accelerated regeneration by latex membranes from Hevea brasiliensis and Hancornia speciosa

Bone loss is a common problem after accidental traumas, cancers, congenital defects, and surgical procedures. The techniques normally used in large bone restoration involve complex and invasive procedures such as grafting. Thus, it is of interest to develop alternatives such as bioactive materials to induce accelerated bone regeneration. Natural rubber (NR) membranes are potential candidates due to their characteristics such as biocompatibility, angiogenic potential, flexibility, mechanical stability, surface porosity, and permeability. The present study aims at assessing the osteogenic potential of NR membranes of clones of high bioactivity of Hevea brasiliensis (RRIM 600 and IAN 873) and of Hancornia speciosa, as well as Physicochemical characterization at the NR membranes by scanning electron microscopy, Fourier transform infrared spectroscopy, and tensile tests. Critical-size bone defects were surgically made in adult male rabbit calvarium. A fibrin sealant (FS) was used to fix the membranes as a replacement for cyanoacrylate. We compared the respective osteogenic potentials of the tested membranes against a control group in healthy animals. The new bone formed was characterized using radiography, x-ray tomography, and histological and morphometric studies. Our results show that both membranes have great potential for regenerating bone tissue, with higher bioactivity compared to the gold standard (PTFE), which was used as positive control. In both NR membranes the stress–strain profile shows low stress at small strain, characteristic of elastomer with a low degree of reticulation, followed by an increase in the stress at high deformation and two main differences between both NRL biomembranes, related to its composition. The FS acted satisfactorily in the tests, being highly recommended as a substitute for cyanoacrylate in this type of application.

Physicochemical/photophysical characterization and angiogenic properties of Curcuma longa essential oil

This study analyzed the physicochemical and photophysical properties of essential oil of Curcuma longa and its angiogenic potential. The results showed that curcumin is the main fluorescent component present in the oil, although the amount is relatively small. The experimental chorioallantoic membrane model was used to evaluate angiogenic activity, showing a significant increase in the vascular network of Curcuma longa and positive control groups when compared to the neutral and inhibitor controls (P <0.05), but no significant difference was found between Curcuma longa essential oil and the positive control (P >0.05). Histological analysis showed extensive neovascularization, hyperemia and inflammation in the positive control group and Curcuma longa when compared to other controls (P <0.05), characteristic factors of the angiogenesis process. In conclusion, Curcuma longa oil showed considerable proangiogenic activity and could be a potential compound in medical applications.

Antigenotoxicity protection of Carapa guianensis oil against mitomycin C and cyclophosphamide in mouse bone marrow

The aim of this study was to evaluate the possible protective of C. guianensis oil against MMC and CP, which are direct- and indirect-acting chemical mutagens, using the micronucleus test. Three experiments were performed. First the C. guianensis oil was co-administered to mice at doses of 250, 500 and 1000 mg/kg bw with 4 mg/kg bw MMC or 50 mg/kg bw CP. Second, the mutagenic drug (CP) was administered ip 50 mg/kg bw and after 6 and 12 hours 250 and 500 mg/kg bw of C. guianensis oil were administered. In the last, C. guianensis oil was administrated (250 and 500 mg/kg bw) during five days and after it was administered ip 50 mg/kg bw CP. The results obtained showed that the C. guianensis oil is not cytotoxic neither genotoxic to mouse bone marrow. Regarding the antimutagenic effect, all doses of C. guianensis oil were significantly (p < 0.05) effective in reducing the frequency of micronucleated polychromatic erythrocytes, when compared with MMC or CP alone. Based on these results, our results suggest that the C. guianensis oil shows medicinal potential as an antimutagenic agent, modulating the mutagenicity caused by both direct- and indirect-acting chemical mutagens, in a mammalian model.

STATE OF THE ART OF SCIENTIFIC LITERATURE ON Hancornia speciosa: TRENDS AND GAPS

O interesse economico e cientifico sobre Hancornia speciosa (mangabeira) vem crescendo nos ultimos anos, devido principalmente a comercializacao de seu fruto e extracao de compostos naturais com alto potencial farmacologico. Neste estudo, foi realizado um levantamento cienciometrico sobre a mangabeira, com o objetivo de fomentar e direcionar futuras pesquisas sobre a especie. Como resultado, verificou-se a baixa produtividade cientifica a respeito da especie, com a publicacao de apenas 131 artigos nos ultimos 69 anos. Alem disso, o presente estudo identificou algumas tendencias na producao bibliografica sobre a mangabeira, com destaque para: o aumento do numero de artigos ao longo dos ultimos anos; o fato de a divulgacao cientifica ser realizada principalmente em revistas de âmbito nacional; a abordagem experimental ser mais frequente do que a abordagem teorico-descritiva; os trabalhos experimentais serem geralmente associados as populacoes de distribuicao geografica restrita; e a producao cientifica ser proveniente, principalmente, de Instituicoes de Ensino Superior. Por outro lado, este estudo tambem permitiu a identificacao de algumas lacunas no conhecimento sobre a mangabeira, entre as quais se destacam: a dificuldade em descrever e caracterizar as linhagens botânicas; a falta de analise da diversidade genetica de populacoes de ampla distribuicao geografica; a carencia de projetos de manejo e de conservacao da especie; a falta de descricao de tecnicas de cultivo, coleta e conservacao de frutos; e a ausencia da identificacao dos compostos naturais responsaveis pela atividade de farmacogica. Espera-se que os dados gerados nesta pesquisa possam ser usados para direcionar futuros estudos sobre a planta.

Intraspecific differentiation of Hancornia speciosa revealed by simple sequence repeat and random amplified polymorphic DNA markers.

Hancornia speciosa, popularly known as mangabeira, is a fruit tree native to the Brazilian Cerrado that shows great economic potential, due to its multiple uses. Intraspecific classification of this species is difficult because it shows high morphological diversity. An early study of the species reported that there are six botanic varieties that differ morphologically mainly in the shapes of their leaves and flowers. Except to note the wide morphological variation and economic potential of this species, few studies have been published about the genetic diversity of mangabeira. Knowledge of the genetic variability of this species among populations would be useful for genetic conservation and breeding programs. Therefore, we tested the transferability of 12 simple sequence repeats from expressed sequence tags (EST-SSRs) from Catharanthus roseus to H. speciosa and used 10 random amplified polymorphic DNA markers to evaluate the genetic variability among botanical varieties of H. speciosa. We obtained a high transferability frequency of EST-SSR markers from C. roseus to H. speciosa (75%). However, EST-SSR markers showed low heterozygosity and locus variability (two or three alleles by locus), which suggest low genetic diversity in the mangabeira samples. The Jaccard dissimilarity index and an examination of geographic distances indicated a non-spatial structuring of the genetic variability. Our markers were unable to distinguish H. speciosa botanical varieties.