Um dos graves obstáculos para a manutenção e estabilidade da produção nacional de arroz (Oryza sativa) reside na susceptibilidade dos genótipos existentes a estresses abióticos. Tendo em vista a importância social e econômica do arroz e os efeitos extremamente danosos desses estresses sobre a agricultura, o conhecimento detalhado das interações entre os estresses abióticos e as respostas dos vegetais frente a esses estímulos ambientais faz-se necessário. O alumínio (Al) é considerado um dos principais fatores limitantes na produção agrícola, inibindo o crescimento das raízes e a absorção de minerais. A toxicidade do Al em plantas ocorre pela sua solubilização em solos com pH baixo ou solos ácidos. Os genes ASR (ABA, Stress and Ripening) são induzidos por estresse e ácido abscísico (ABA) em plantas, e seus níveis de expressão são rapidamente aumentados em resposta à salinidade e seca. Recentemente, foi demonstrado que o gene que codifica a proteína ASR5 é responsivo ao Al em raízes de arroz. Apesar do arroz ser considerado um dos cereais mais resistentes a Al, os mecanismos básicos de tolerância a este metal são pouco conhecidos no arroz em comparação a outros cereais. Por meio do presente trabalho objetivamos: i) a caracterização funcional dos membros da família gênica ASR de arroz em reposta ao Al; e ii) a construção de vetores binários de transformação de plantas visando o estudo da localização subcelular da proteína codificada pelo gene OsASR5, e o silenciamento gênico da família ASR de arroz. As análises dos transcritos por qRT-PCR mostraram que todos os genes da família ASR de arroz ssp Japonica respondem ao Al. Por outro lado, OsASR5 não sofre modulação de sua expressão em resposta ao Al em raízes de arroz ssp Indica. Essas diferenças de respostas dos genes OsASR5 em distintas variedades pode refletir diferenças no grau de tolerância ao Al de cada um desses genótipos. / One of the major obstacles to maintain the stability of the national production of rice (Oryza sativa) lies on the susceptibility of the different genotypes to abiotic stress. In view of the social and economic importance of rice and due to the extremely harmful effects of stress in agriculture, detailed knowledge of the interactions between these stresses and plant responses to environmental stimuli is necessary. Aluminum (Al) is considered one of the main limitation factors for agricultural productivity, inhibiting root growth and mineral absorption. Al toxicity occurs by its solubilization in soils with low pH or acid soils. The ASR (ABA, Stress and Ripening) genes are induced by stress and abscisic acid (ABA) in plants, and their expression levels are quickly increased in response to salinity and drought. Recently, it was demonstrated that the ASR5 gene is responsive to Al in rice roots. Despite the fact that rice is considered one of the most resistant crops to Al, the basic mechanisms of tolerance to this metal are poorly known when compared to other crops. This study aimed the functional characterization of the gene expression of rice ASR family members in response to Al, and the construction of binary vectors for the subcellular localization of the protein codified by the OsASR5 gene, and the construction of a gene silencing binary vector for the ASR family. Analyses of transcripts by qRT-PCR showed that in the ssp Japonica, all ASR genes responded to Al. In contrast, OsASR5 do not suffer expression modulation in response to Al in rice roots of ssp Indica. These differences in response of the OsASR5 gene in distinct varieties may reflect differences in the degree of Al tolerance in each genotype.

Resumo: Produtos cárneos que contenham propriedades organolépticas específicas e que atendam às exigências dos consumidores, são cada vez mais valorizados no mercado. Apesar da sua importância, as características de qualidade da carne em geral não são consideradas em programas de melhoramento genético animal, em razão do alto custo e dificuldade de mensuração. Marcadores moleculares identificados em análises genômicas, têm sido estudados em rebanhos Nelore. Trabalhos que visam o entendimento da expressão de genes e proteínas envolvidos na determinação de características relacionadas a gordura intramuscular da carne, são realizados com o intuito de auxiliar no entendimento da expressão dos genes relacionados a possíveis biomarcadores. Assim, inúmeros trabalhos que visam avaliar a expressão diferencial de genes podem ser encontrados na literatura, no entanto, devido a divergências entre o manejo e as raças estudadas, os resultados são discordantes, e a validação dos genes mais comumente encontrados, se faz necessário para que essas informações possam ser úteis em programas de melhoramento genético. Assim, este estudo teve como finalidade buscar na literatura genes diferencialmente expressos associados à característica de deposição de gordura intramuscular, avaliada por meio de duas técnicas, e validá-los em uma população de bovinos Nelore com fenótipo extremo para a característica gordura intramuscular por meio da técnica de PCR quantitativa em tempo real. Além disto, foi verificado se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Meat products with specific organoleptic properties and that attend the requirements of consumers, are increasingly valued in the market. Despite its importance, meat quality traits have not been widely used in animal breeding programs because of the high cost and difficulty of measurement. Molecular markers identified in genomic analyzes have been studied in Nellore herds. Studies aimed at understanding the expression of genes and proteins involved in the determination of characteristics such as intramuscular fat from meat are performed with the aim of helping to understand the expression of the genes related to possible biomarkers. Several studies that aim the expression of differentially expressed genes can be found in the literature, however, because of differences between management and breeds studied the results are discordant, and the validation of the common genes found is necessary for this information to be useful in breeding programs. Thus, this study aimed to search the literature for differentially expressed genes related to the marbling trait, by means of the two techniques for the evaluation of this trait and to validate them in a commercial herd of Nellore cattle, by quantitative real time PCR techniques. Besides that, this study will verify if the differentially expressed genes are being translated into differentially expressed proteins, using advanced mass spectrometry technique (LC-MS/MS). For this, 24 divergent samples were selected for each intramuscular ... (Complete abstract click electronic access below) / Mestre

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Non-specific lipid transfer proteins is a large and diverse protein family found in plants, with roles in biological systems ranging from long distance signaling to plant pathogen defense. Little is known about the roles of nsLTPs, but recent studies have cast some light on the issue, among other things proposing that they may be involved in the cutice formation on land-living liverworts, mosses and non-seedbearing plants. Increased cuticle formation is thought to be a part of a plants defense system against stress. In this experiment, the expression of nsLTPs type G in the moss Physcomitrella patens was examined by qRT-PCR on cDNA synthesized from already existing mRNA samples from moss under different abiotic stresses. The different stresses were UV-light, salt (ion toxicity), heavy metal, cold drought, plant hormone and osmosis. House-keeping gene P. patens beta-tubuline 1 was used as reference and relative expression analysis was performed. The study showed a general down-regulation of PpLTPg's in the abiotically stressed samples, and the possible coupled regulatory response of PpLTPg3 and PpLTPg5. The results imply that the PpLTPg's in Physcomitrella patens could be connected to biological processes that cease during stress, or that they worl through negative feedback to support plant defense against stress.

During the water-to-land transition, that occurred approximately 450 MYA, novel habitats wererevealed to the emerging plants. This terrestrial habitat was a harsh environment compared to theaquatic, with shifting substrate content, irregular supply of water, damaging UV-radiation andrapid fluctuating temperatures. Non-specific lipid transfer proteins (nsLTP) are today only foundin the land living plants and not in the green algae. This suggests that these genes might haveevolved to help the plants cope with the stressful conditions. In this study the expression patternhas been analysed of the nsLTPs in the moss Physcomitrella patens during the possible conditionsthat raised during the water-to-land transition. The moss was exposed to salt, UV-B, drought, copper, cold and osmotic stress. Quantitative real-time PCR was used to analyse the transcription levels. I found that six genes were upregulated during either cold, dehydration or UV-B stress. This suggest that these genes are involved in the plant defense against these abiotic stresse

Two species of bees in the genus Apis, real honey bees, has long been of interest for man. These two are the European honey bee, Apis mellifera, and the Asian honey bee, Apis cerana. In Vietnam, beekeeping is of great importance, both with A.cerana and A.mellifera. The aim of this project was to investigate if the introduction of the European honey bee in Asia has affected the Asian honey bee, and whether different pathogens from A.mellifera have been transferred to A.cerana. Totally 40 samples, 20 from every species, were analysed for 8 different viruses. RNA was extracted and analysed with qRT-PCR. The results showed that 5 different viruses were present in the samples, DWV, CBPV, BQCV, SPV and SBV. SPV and SBV were only found occasionally while DWV, CBPV and BQCV were present in the majority of the samples. Differences in virus titres between the two bee species were significant for CBPV and BQCV, however the result for DWV titres was not considered significant. DWV therefore seem to be a ubiquitous virus in Vietnamese beekeeping irrespective of species. Further, the results cannot describe the influence or origin of the viruses but only confirm their presence. Additional investigations are needed in order to answer this question.

Wheat is the most important grain crop grown in our country providing greatest part of the daily nutritional requirement. Abiotic factors including salinity, drought, cold and heat stresses affect quality and yield of wheat varieties used for the production of both bread and pasta flour. NAC proteins form one of the widest families of plant specific transcription factors. Members of this family are related with development, defense and abiotic stress responses. TaNAC69-1 and TtNAM-B2 genes were isolated from T.aestivum and T.turgidum, respectively. Then they were cloned into different monocot and dicot expression vectors to be used for further wheat and tobacco genetic transformation studies. To understand effects of salinity, drought, cold and heat stresses on expression profiles of TaNAC69-1 and TtNAM-B2 genes, quantitative real time PCR was performed. The time series expression profiles of TaNAC69-1 show that it was signi

Streptomyces clavuligerus produces industrially important secondary metabolites such as cephamycin C (a &beta / -lactam antibiotic) and clavulanic acid (a potent &beta / -lactamase inhibitor). Cephamycin C is active against penicillin-resistant bacteria due to presence of methoxyl group in C-7 position of cephalosporin nucleus. Clavulanic acid is prescribed in combination with &beta / -lactams for treatment of various bacterial infections. Cephamycin C and clavulanic acid gene clusters form &beta / -lactam supercluster in S. clavuligerus genome. CcaR (Cephamycin C-Clavulanic Acid Regulator), encoded by ccaR, located in cephamycin C gene cluster, is a positive regulator of &beta / -lactam supercluster. Previous studies on cephamycin C gene cluster have used different techniques, such as S1 nuclease (Paradkar et al., 1994), Northern blot (Perez-Llarena et al., 1997), and Western blot (Alexander and Jensen, 1998) to determine expression of cephamycin C genes at mRNA level and to identify their functions at protein level, and they have studied on different parts of the cluster. Hence, a comprehensive study is needed to understand molecular mechanisms of pathway-specific regulation of cephamycin C production by S. clavuligerus. In this study, time-dependent expression levels of cephamycin C gene cluster in a ccaR-disrupted mutant and ccaR-overexpressed recombinant strain of S. clavuligerus as compared to those in the wild strain were analysed by RT-PCR and qRT-PCR. In addition, DNA-binding sequences of CcaR on cephamycin C gene cluster were examined by EMSA. The effect of ccaR disruption and overexpression on cephamycin C and clavulanic acid yields were determined by bioassay and HPLC. Three polycistronic and two monocistronic transcripts were obtained by RT-PCR. CcaR regulation showed its effect on mostly ccaR, lat, cmcI, cefD, blp and cefF expression levels. qRT-PCR data was supported by EMSA showing CcaR binding to lat, cefD&ndash / cmcI and ccaR promoters. ccaR overexpression from multi-copy recombinant plasmid resulted in significant increase in cephamycin C and clavulanic acid yields, making the respective recombinant strain as an attractive industrial strain. qRT-PCR data presented herein constitute the first that reveal the effect of CcaR activator on the expression of cephamycin C genes in a time-dependent manner.

The marine diatom genus Pseudo-nitzschia includes species that produce domoic acid, a neurotoxin responsible for illness and mortality in both humans and marine wildlife. Because of the expertise and time required for the microscopic discrimination of species, molecular methods that monitor environmental concentrations of Pseudo-nitzschia provide a rapid alternative for the early detection of blooms and prediction of toxin accumulation. We have developed a nucleic acid sequence-based amplification with internal control RNA (IC-NASBA) assay and a quantitative reverse transcription PCR (qRT-PCR) assay for the detection of the toxic species P. multiseries targeting the ribulose- 1,5-biphosphate carboxylase/oxygenase small subunit (rbcS) gene. Both methods use RNA amplification and fluorescence-based real-time detection. Due to a limited rbcS sequence database, primers were designed and used to sequence this gene from 14 strains of Pseudo-nitzschia (including four P. multiseries) and 19 other marine diatoms. The IC-NASBA and qRT-PCR assays had a limit of detection of one cultured cell of P. multiseries and were linear over four and five orders of magnitude, respectively (r2 ! 0.98). Neither of the assays detected closely related organisms outside the Pseudo-nitzschia genus, and the qRT-PCR assay was specific to P. multiseries. While cross-reactivity of primers with unknown species prevented reliable detection of P. multiseries in spiked environmental samples using IC-NASBA, the qRT-PCR assay had positive detection from 107 cells/L to 103 cells/L. Nearly a 1:1 relationship was observed between predicted and calculated cell concentrations using qRT-PCR. Based on a diel expression study, the rbcS transcript copy number per cell ranged from 2.16 x 104 to 5.35 x 104, with the highest expression during early to mid photoperiod. The rbcS qRT-PCR assay is useful for the detection and enumeration of low concentrations of P. multiseries in the environment.