Otimização de marcadores desenvolvidos para PCR convencional para uso em qPCR visando a diagnose de Xanthomonas campestris pv viticola em videira.

No Brasil, Xanthomonas campestris pv. viticola (Xcv), causadora do cancro bacteriano em videira, é uma praga quarentenária A2, com ocorrência no Semiárido Nordestino. A bactéria pode ser disseminada de plantas assintomáticas pela distribuição de material propagativo e ocorrências restritas da doença em outras regiões foram identificadas. Para diagnose confiável por PCR convencional, o DNA deve ser extraído de culturas de bactérias isoladas de tecido com sintomas suspeitos. Com a técnica, é possível detectar até 0,25 pg de DNA bacteriano total. Atualmente, métodos que empregam tecidos assintomáticos não estão disponíveis. O objetivo deste trabalho foi desenvolver um protocolo sensível à detecção de Xcv por qPCR, empregando iniciadores disponíveis da técnica convencional.

Multiplex PCR para a identificação de cochonilhas farinhentas (Hemiptera Pseudococcidae) presentes na cultura da videira no Brasil.

Uma alta diversidade de espécies de cochonilhas farinhentas (Hemiptera:Pseudococcidae) tem sido observada na cultura da videira. Algumas espécies são inclusive consideradas pragas quarentenárias para outros países. A identificação de cochonilhas farinhentas é um dos fatores limitantes para o manejo destes insetos no campo, em decorrência da grande similaridade morfológica observada entre espécies próximas e da variação morfológica que ocorre em alguns grupos em decorrência do hospedeiro e da temperatura de desenvolvimento. Além disto, a identificação destes insetos baseia-se em características morfológicas presentes apenas em fêmeas adultas, realizada por poucos especialistas. O objetivo deste trabalho foi desenvolver um ?kit? de identificação molecular das principais espécies de cochonilhas farinhentas presentes na cultura da videira no Brasil [(Dysmicoccus brevipes (Cockerell), Phenacoccus solenopsis (Tinsley), Planococcus citri (Risso), Planococcus ficus (Signoret) e Pseudococcus viburni (Signoret)]. Cochonilhas farinhentas foram coletadas na Serra Gaúcha, RS, Vale do São Francisco (Polos Juazeiro- BA e Petrolina-PE) e em cidades produtoras de uvas do Paraná.

Processamento de liquido cefalo-raquidiano para extração de RNA genômico do vírus da artrite-encefalite caprina e diagnóstico molecular por RT-nested PCR.

Este comunicado descreve os procedimentos de coleta, processamento do líquido céfalo-raquidiano, para a extração de RNA genômico viral, e de detecção do vírus através da técnica de RT-nested PCR, potencial método de diagnóstico molecular da CAE.

Processamento de sangue e líquido sinovial para extração de RNA genomico do vírus da artrite-encefalite caprina e diagnóstico molecular por RT-nested PCR.

Este comunicado descreve os procedimentos de coleta e processamento de líquido sinovial e sangue seguido pela extração de RNA genômico e, finalmente, o diagnóstico molecular do vírus pela técnica de RT-nested PCR.

Processamento de sêmen para extração de RNA genômico do vírus da artrite-encefalite caprina e diagnóstico molecular por RT-nested PCR.

O presente comunicado descreve os procedimentos necessários para a coleta e processamento de amostras de sêmen de caprinos infectados pelo CAEV para posterior extração do RNA viral por meio de um método baseado em centrifugação em coluna de sílica. A avaliação da presença de RNA no sêmen será feita, diretamente, por meio da reação de RT-nested PCR, portencial método de diagnóstico molecular da CAE.

Occurrence of apple stem grooving capillovirus in Santa Catarina, Brazil, detected by RT-PCR.

1999

Diagnóstico molecular por PCR-RFLP e gene LYS2 para identificação de Fusarium decemcellulare.

Fusarium decemcellulare é encontrado como agente causal de doenças com diferentes sintomas em diversas espécies de plantas em regiões tropicais e subtropicais. Em guaranazeiro, espécie nativa da Amazônia de importância econômica e social, a doença denominada de complexo superbrotamento é atualmente um dos principais problemas da cultura. Técnicas moleculares são cada vez mais requeridas para identificação rápida e segura de patógenos como complemento as técnicas convencionais. O objetivo do trabalho foi desenvolver métodos moleculares por PCR e PCR-RFLP para rápida identificação de F. decemcellulare.

Caracterização de Fusarium decemcellulare isolado de guaranazeiro (Paullinia cupana var. sorbilis) por meio de ERIC-PCR.

A presente pesquisa visa analisar a diversidade genética De F. decemcellulare isolado de mudas e plantas adultas de guaranazeiro com sintomas de superbrotamento, hipertrofia floral ou galhas por meio do marcador molecular ERIC-PCR.

RAPD em pau-rosa (Aniba rosaeodora Ducke): adaptação do método para coleta de amostras in situ, ajuste das condições de PCR e apresentação de um processo para selecionar bandas reprodutíveis.

Os objetivos deste trabalho foram: (1) validar um método para a coleta de material vegetal de pau-rosa; (2) selecionar um método para a extração de DNA de folhas de pau-rosa, em quantidade e com qualidade adequadas para a obtenção de padrões RAPD e (3) desenvolver e validar um critério, baseado no grau de reprodutibilidade para selecionar bandas RAPD para as análises genéticas.

Detecção de oito vírus em videiras por meio de RT-PCR em tempo real.

2011

The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

BACKGROUND: The murine ghrelin gene (Ghrl), originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0). We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. -----FINDINGS: 5' RACE revealed two transcription start sites (TSSs) in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR), we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. -----CONCLUSION: We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1) raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

Bridging technical and HCI research : creating usable ubiquitous computing

This paper describes methods used to support collaboration and communication between practitioners, designers and engineers when designing ubiquitous computing systems. We tested methods such as “Wizard of Oz” and design games in a real domain, the dental surgery, in an attempt to create a system that is: affordable; minimally disruptive of the natural flow of work; and improves human-computer interaction. In doing so we found that such activities allowed the practitioners to be on a ‘level playing ground’ with designers and engineers. The findings we present suggest that dentists are willing to engage in detailed exploration and constructive critique of technical design possibilities if the design ideas and prototypes are presented in the context of their work practice and are of a resolution and relevance that allow them to jointly explore and question with the design time. This paper is an extension of a short paper submitted to the Participatory Design Conference, 2004.

Implication of 3D culture system for study of prostate cancer (CaP) mediated bone metastasis

Introduction : For the past decade, three dimensional (3D) culture has served as a foundation for regenerative medicine study. With an increasing awareness of the importance of cell-cell and cell-extracellular matrix interactions which are lacking in 2D culture system, 3D culture system has been employed for many other applications namely cancer research. Through development of various biomaterials and utilization of tissue engineering technology, many in vivo physiological responses are now better understood. The cellular and molecular communication of cancer cells and their microenvironment, for instance can be studied in vitro in 3D culture system without relying on animal models alone. Predilection of prostate cancer (CaP) to bone remains obscure due to the complexity of the mechanisms and lack of proper model for the studies. In this study, we aim to investigate the interaction between CaP cells and osteoblasts simulating the natural bone metastasis. We also further investigate the invasiveness of CaP cells and response of androgen sensitve CaP cells, LNCaP to synthetic androgen.----- Method : Human osteoblast (hOB) scaffolds were prepared by seeding hOB on medical grade polycaprolactone-tricalcium phosphate (mPLC-TCP) scaffolds and induced to produce bone matrix. CaP cell lines namely wild type PC3 (PC3-N), overexpressed prostate specific antigen PC3 (PC3k3s5) and LNCaP were seeded on hOB scaffolds as co-cultures. Morphology of cells was examined by Phalloidin-DAPI and SEM imaging. Gelatin zymography was performed on the 48 hours conditioned media (CM) from co-cultures to determine matrix metalloproteinase (MMP) activity. Gene expression of hOB/LNCaP co-cultures which were treated for 48 hours with 1nM synthetic androgen R1881 were analysed by quantitative real time PCR (qRT-PCR).----- Results : Co-culture of PCC/hOB revealed that the morphology of PCCs on the tissue engineered bone matrix varied from homogenous to heterogenous clusters. Enzymatically inactive pro-MMP2 was detected in CM from hOBs and PCCs cultured on scaffolds. Elevation in MMP9 activity was found only in hOB/PC3N co-culture. hOB/LNCaP co-culture showed increase in expression of key enzymes associated with steroid production which also corresponded to an increase in prostate specific antigen (PSA) and MMP9.----- Conclusions : Upregulation of MMP9 indicates involvement of ECM degradation during cancer invasion and bone metastases. Expression of enzymes involved in CaP progression, PSA, which is not expressed in osteoblasts, demonstrates that crosstalk between PCCs and osteoblasts may play a part in the aggressiveness of CaP. The presence of steroidogenic enzymes, particularly, RDH5, in osteoblasts and stimulated expression in co-culture, may indicate osteoblast production of potent androgens, fuelling cancer cell proliferation. Based on these results, this practical 3D culture system may provide greater understanding into CaP mediated bone metastasis. This allows the role of the CaP/hOB interaction with regards to invasive property and steroidogenesis to be further explored.

Multilineage differentiation potential of bone and cartilage cells derived from explant culture

To date, mesenchymal stem cells (MSCs) from various tissues have been reported, but the yield and differentiation potential of different tissue-derived MSCs is still not clear. This study was undertaken in an attempt to investigate the multilineage stem cell potential of bone and cartilage explant cultures in comparison with bone marrow derived mesenchymal stem cells (BMSCs). The results showed that the surface antigen expression of tissue-derived cells was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing markers related to adhesion (CD29, CD166) and stem cells (CD90, CD105). The tissue-derived cells were able to differentiate into osteoblast, chondrocyte and adipocyte lineage pathways when stimulated in the appropriate differentiating conditions. However, compared with BMSCs, tissue-derived cells showed less capacity for multilineage differentiation when the level of differentiation was assessed in monolayer culture by analysing the expression of tissue-specific genes by reverse transcription polymerase chain reaction (RT-PCR) and histology. In high density pellet cultures, tissue-derived cells were able to differentiate into chondrocytes, expressing chondrocyte markers such as proteoglycans, type II collagen and aggrecan. Taken together, these results indicate that cells derived from tissue explant cultures reserved certain degree of differentiation properties of MSCs in vitro.