Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBP beta transcriptional activity

Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBP beta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPAR gamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPAR gamma (60%), C/EBP alpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBP alpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBP beta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBP alpha promoter. However, when treated with melatonin, the level of C/EBP alpha promoter activation by C/EBP beta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBP beta.

A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010; 107: 469-477. (C) 2010 Wiley Periodicals, Inc.

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS

Phosphoribosyl pyrophosphate synthetase (PRS-EC:2.7.6.1) is an important enzyme present in several metabolic pathways, thus forming a complex family of isoenzymes. However, plant PRS enzymes have not been extensively investigated. In this study, a sugarcane prs gene has been characterized from the Sugar Cane Expressed Sequence Tag Genome Project. This gene contains a 984-bp open reading frame encoding a 328-amino acid protein. The predicted amino acid sequence has 77% and 78% amino acid sequence identity to Arabidopsis thaliana and Spinacia oleracea PRS4, respectively. The assignment of sugarcane PRS as a phosphate-independent PRS isoenzyme (Class II PRS) is verified following enzyme assay and phylogenetic reconstruction of PRS homologues. To gain further insight into the structural framework of the phosphate independence of sugarcane PRS, a molecular model is described. This model reveals the formation of two conserved domains elucidating the structural features involved in sugarcane PRS phosphate independence. The recombinant PRS retains secondary structure elements and a quaternary arrangement consistent with known PRS homologues, based on circular dichroism measurements.

Bioluminescent sensor for naphthalene in air: Cell immobilization and evaluation with a dynamic standard atmosphere generator

A dynamic atmosphere generator with a naphthalene emission source has been constructed and used for the development and evaluation of a bioluminescence sensor based on the bacteria Pseudomonas fluorescens HK44 immobilized in 2% agar gel (101 cell mL(-1)) placed in sampling tubes. A steady naphthalene emission rate (around 7.3 nmol min(-1) at 27 degrees C and 7.4 mLmin(-1) of purified air) was obtained by covering the diffusion unit containing solid naphthalene with a PTFE filter membrane. The time elapsed from gelation of the agar matrix to analyte exposure (""maturation time"") was found relevant for the bioluminescence assays, being most favorable between 1.5 and 3 h. The maximum light emission, observed after 80 min, is dependent on the analyte concentration and the exposure time (evaluated between 5 and 20 min), but not on the flow rate of naphthalene in the sampling tube, over the range of 1.8-7.4 nmol min(-1). A good linear response was obtained between 50 and 260 nmol L-1 with a limit of detection estimated in 20 nmol L-1 far below the recommended threshold limit value for naphthalene in air. (c) 2008 Elsevier B.V. All rights reserved.

Measuring Application Availability,Usage and Performance : Implementation of EnView System

The main objective for this degree project is to implement an Application Availability Monitoring (AAM) system named Softek EnView for Fujitsu Services. The aim of implementing the AAM system is to proactively identify end user performance problems, such as application and site performance, before the actual end users experience them. No matter how well applications and sites are designed and nomatter how well they meet business requirements, they are useless to the end users if the performance is slow and/or unreliable. It is important for the customers to find out whether the end user problems are caused by the network or application malfunction. The Softek EnView was comprised of the following EnView components: Robot, Monitor, Reporter, Collector and Repository. The implemented system, however, is designed to use only some of these EnView elements: Robot, Reporter and depository. Robots can be placed at any key user location and are dedicated to customers, which means that when the number of customers increases, at the sametime the amount of Robots will increase. To make the AAM system ideal for the company to use, it was integrated with Fujitsu Services’ centralised monitoring system, BMC PATROL Enterprise Manager (PEM). That was actually the reason for deciding to drop the EnView Monitor element. After the system was fully implemented, the AAM system was ready for production. Transactions were (and are) written and deployed on Robots to simulate typical end user actions. These transactions are configured to run with certain intervals, which are defined collectively with customers. While they are driven against customers’ applicationsautomatically, transactions collect availability data and response time data all the time. In case of a failure in transactions, the robot immediately quits the transactionand writes detailed information to a log file about what went wrong and which element failed while going through an application. Then an alert is generated by a BMC PATROL Agent based on this data and is sent to the BMC PEM. Fujitsu Services’ monitoring room receives the alert, reacts to it according to the incident management process in ITIL and by alerting system specialists on critical incidents to resolve problems. As a result of the data gathered by the Robots, weekly reports, which contain detailed statistics and trend analyses of ongoing quality of IT services, is provided for the Customers.

Use of microprojectile bombardment in transient expression assays to analyze protochlorophyllide reductase gene expression in greening maize seedling leaf cells

In young cells of leaf meristems the progenitors of chloroplasts are small organelles known as proplastids, which divide and differentiate into chloroplasts. However, in the absence of light, proplastids undergo a different sequence of development and become etioplasts. When light is supplied to etiolated plants during the "greening" process, etioplasts differentiate into chloroplasts containing chlorophyll. An important light dependent step in chlorophyll biosynthesis is the photoreduction of protochlorophyllide to chlorophyllide by the NADPH:protochlorophyllide reductase (PCR) enzyme. This enzyme is present at high activity only in etiolated tissue and during early stages of light-induced chlorophyll synthesis. The enzyme and its corresponding mRNAs decrease dramatically with prolonged exposure to light. We have investigated the light-dependent transcriptional regulation of a PCR gene in greening maize leaf cells using a transient expression assay based on microprojectile bombardment. The promoter region was isolated and cloned into a ?-glucuronidase (GUS) reporter gene expression plasmid. We have used this chimeric plasmid in tungsten particle bombardment of both etiolated and greening maize seedling leaves to determine whether the cloned promoter region contains regulatory sequences that control light-responsive PCR gene expression.

Who Was Elizabeth Hanson? Book reveals the secret circumstances surrounding the death of the CIA's rising star

Book by Washington Post reporter reveals the secret circumstances surrounding the death of rising CIA star Elizabeth Hanson ’02.

Transformação genética em cevada por bombardeamento de partículas

A presente Tese de Doutorado objetivou: (1) definir um método eficiente de transformação genética, por bombardeamento de partículas, para a obtenção de plantas transgênicas de cultivares brasileiras de cevada e (2) identificar gene(s) codificante(s) de quitinase(s) potencialmente capaz(es) de conferir resistência ao fungo patogênico de cevada Bipolaris sorokiniana. Culturas de calos obtidos a partir de escutelos imaturos das cultivares Brasileiras de cevada MN-599 e MN-698 (Cia. de Bebidas das Américas, AMBEV) foram bombardeadas com partículas de tungstênio e avaliadas quanto à expressão do gene repórter gusA através de ensaios histoquímicos de GUS e quanto ao efeito dos bombardeamentos na indução estruturas embriogênicas e regeneração de plantas. As condições de biobalística analisadas incluíram a região promotora regulando a expressão de gusA, tipo e pressão de gás hélio de dois aparelhos de bombardeamento, distância de migração das partículas, número de tiros e a realização de pré e pós-tratamento osmótico dos tecidos-alvo. No presente trabalho foram obtidos um número bastante alto de pontos azuis por calo, a indução de calos embriogênicos e embriões somáticos em uma freqüência de até 58,3% e a regeneração de 60 plantas, sendo 43 de calos bombardeados. As melhores condições observadas foram o promotor e primeiro íntron do gene Adh de milho (plasmídeo pNGI), o aparelho de bombardeamento “ Particle Inflow Gun” (PIG) utilizando-se a distância de migração de partículas de 14,8 cm, dois tiros disparados por placa e a realização de tratamento osmótico dos explantes com 0,2 M de manitol e 0,2 M de sorbitol 4-5 horas antes e 17-19 horas depois dos bombardeamentos. Das 43 plantas obtidas de calos bombardeadas, 3 apresentaram atividade de GUS na base das suas folhas. A utilização de primers sintéticos definidos a partir de genes de quitinases descritos na literatura em PCRs resultou na amplificação de dois fragmentos de aproximadamente 700 e 500 pb a partir de DNA total das cvs. MN-599 e MN-698 de cevada e um fragmento, com aproximadamente 500 pb, a partir do DNA total do isolado A4c de Trichoderma sp. Estes fragmentos foram purificados dos géis de agarose e diretamente seqüenciados de forma manual e automática. Os fragmentos de 700 e 500 pb amplificados do genoma da cultivar MN-599 foram identificados como genes de quitinases de cevada e o fragmento de 500 pb do isolado A4c de Trichoderma sp. não apresentou homologia com seqüências conhecidas de quitinases depositadas no EMBL/GenBank. A utilização de novos pares de primers, representando seqüências conservadas de quitinases do fungo Metarhizium anisopliae, resultou na amplificação de 3 fragmentos a partir do DNA total do isolado A4b de Trichoderma sp., que estão sendo purificados para realização de seqüenciamento.

Políticas públicas para erradicação do trabalho escravo contemporâneo no Brasil: um estudo sobre a dinâmica das relações entre os atores governamentais e não-governamentais

O estudo tem como objetivo identificar na evolução das políticas públicas para erradicação do trabalho escravo os diferentes atores e a dinâmica das relações entre eles. A ocorrência da escravidão contemporânea pôde se dar a partir da contribuição de alguns fatores estruturais e conjunturais, tais como o processo de aprofundamento do capitalismo e de modernização conservadora no país e especificamente na agricultura e relações políticas, sociais e históricas que perpetuam a enorme concentração fundiária brasileira. Além disso, algumas relações pessoais, sociais e políticas de intermediação de interesses entre Estado e sociedade, tais como clientelismo e patronagem e redes de políticas, de modo geral e de forma mais específica nas políticas agrárias, também interferem no desenvolvimento dos processos de políticas públicas e dentre elas nas políticas de combate ao trabalho escravo. Desse modo, a dissertação tem como problema a investigação da dinâmica das relações entre atores governamentais e nãogovernamentais na formulação e implantação das políticas públicas de erradicação ao trabalho escravo no Brasil. Para tanto, o estudo foi realizado por meio de pesquisa bibliográfica, documental e de campo, tendo entrevistado os seguintes atores políticos: MTE, MPT, OIT, CPT, ONG Repórter Brasil, GPTEC e OAB. Os dados foram analisados pelo método de análise de conteúdo, sob um viés qualitativo. Os resultados da pesquisa permitiram identificar a formação de múltiplas redes entre os atores governamentais e não-governamentais envolvidos nesta questão, demonstrando certa divisão entre as redes que atuam lutando pelo combate ao trabalho escravo e outras que se posicionam como uma certa resistência a esse combate, devido a interesses econômicos e políticos, revelando, assim, um jogo de forças que ora apresenta avanços e conquistas, ora mostra retrocessos ou estagnação na luta contra a escravidão contemporânea brasileira.

Avaliação da eficiência de co-transformação de soja (Glycine max (L.) Merrill) via bombardeamento de partículas utilizando um gene de seleção e um gene de interesse em plamídeos diferentes

Visando aumentar a resistência a moléstias fúngicas, o presente trabalho teve como objetivo introduzir um gene (chit1) que codifica uma quitinase do fungo Metarhizium anisopliae em cultivares de soja [Glycine max (L.) Merrill]. A co-transformação foi a estratégia escolhida, visando a obtenção de plantas livres de transgenes marcadores na progênie das plantas transformadas. A co-transformação foi realizada via biolística, tendo como tecido-alvo conjuntos de embriões somáticos globulares das cultivares MG/BR46 Conquista e IAS-5. O plasmídeo pGusHyg, que contém o gene repórter gusA e o gene marcador hpt, foi bombardeado concomitantemente com o plasmídeo pMOG463chit1, que porta o gene chit1. Os conjuntos de embriões bombardeados foram transferidos para meio seletivo contendo higromicina, visando a obtenção de material estavelmente transformado. Os conjuntos embriogênicos higromicina-resistentes foram transferidos seqüencialmente para meios de proliferação D-20 (sem higromicina), maturação e regeneração. No total, foram obtidos 387 e 380 embriões histodiferenciados das cultivares MG/BR46 Conquista e IAS-5, respectivamente. Plantas transgênicas adultas e férteis foram regeneradas. Para avaliar a eficiência da estratégia de cotransformação, foram realizadas análises moleculares de embriões histodiferenciados e de plantas regeneradas. Os resultados obtidos neste trabalho permitiram o cálculo da taxa de co-transformação de 44% para os embriões histodiferenciados da cultivar MG/BR46 Conquista e de 50% para plantas de IAS-5. Não existem, até o momento, relatos de trabalhos em soja utilizando embriões somáticos globulares em proliferação como alvo para estudos de co-transformação.

Profissão: repórter esportivo - das narrativas da nação ao ofício do jornalista contemporâneo

Essa dissertação tem por objetivo investigar de que maneira o repórter esportivo contemporâneo se relaciona com o futebol e como essa relação interfere no seu ofício e na construção do texto jornalístico. Mais especificamente, busca identificar as práticas adotadas e as estratégias narrativas utilizadas na construção de seus textos. O estudo dos elementos presentes na prática jornalística tem como perspectiva que a mídia participou ativamente da construção do imaginário futebolístico nacional e é na contemporaneidade a grande mediadora entre o público e os eventos esportivos. É sob este prisma que se pretende apurar como se dá a relação com o trabalho do profissional de imprensa que se identifica com seu objeto, analisar qual o lugar da emoção na composição da notícia esportiva e verificar que valores imbuem esse profissional na construção do texto jornalístico. A análise tem como corpus entrevistas de história oral temática com seis jornalistas que atuam ou atuaram como repórteres esportivos em jornais diários da cidade do Rio de Janeiro, todos com expressiva trajetória no meio esportivo, o que lhes confere credibilidade e autoridade públicas para protagonizar a constituição da memória da imprensa esportiva brasileira. O trabalho é constituído por três etapas: 1) a contextualização – quando se relaciona futebol e formação social brasileira, imprensa e jornalismo esportivo -; 2) o estudo do jornalismo como ofício a partir das teorias da Sociologia das Profissões e das estratégias narrativas utilizadas no texto noticioso esportivo nos jornais; e 3) a análise qualitativa das entrevistas temáticas.

Business diplomacy: the compass rose of foreign markets

With transnational corporations (TNCs) around the world today numbering over 60,000 and more than 800,000 affiliates working abroad, it is easy to understand how modern day international business could have transformed into a major global player serving at the axis of politics, social and environmental responsibility. Additionally, with accountability to a large variety of both public and private stakeholders, all exerting significant power and influence, today’s global corporate structure is reinventing modern international relations, and in some cases, dominating it. (Muldoon 2005) This transformative nature of globalization today can also serve as a source of friction among this growing chorus of players and is bringing irreversible change to these relationships and how they impact and influence business around the world. (Muldoon 2005) From the largest to the smallest international corporation seeking to expand into new international markets, the challenges that come with corporate ambition can mean the difference between success and failure and they find a home at the intersection of international relations, diplomacy and economics. To successfully navigate these challenges, especially in emerging economies, a company must now factor in more than just the “bottom line” and address complex issues that include human rights differences, environmental regulations, labor rights and values of each country. (Henisz, 2014) Combined with modern-day mobility achieved through technology and the Internet, corporations today have a great capacity to reach targeted audiences and establish a presence, but it is this same technology that also allows for immediate response to any corporate action. This constant, 24-hour news cycle, where everyone is made to be a real-time reporter through social media, has created a situation that demonstrably necessitates the ability to not only 3 respond immediately, but also to have real-time understanding of the challenges faced by a corporation as it looks toward global expansion. International Business Diplomacy, or simply Business Diplomacy as it will be referred to in this paper, combines all of these nuanced factors into a relatively new discipline that offers companies looking to expand into new markets, guidelines and directives so that they can more strategically map corporate direction, limit risk and achieve their objectives. This paper will examine the history of diplomacy and how the concept of statecraft became intertwined with the increasing globalization of business. Following a scholarly examination of how modern Business Diplomacy came into being, and the unique challenges that come with its application, particularly the liabilities needed to be overcome, this paper will apply the concept to the Brazilian aerospace manufacturer Embraer, tracking its strategic emergence from a small, regionally focused aircraft producer to global leader in the regional and executive jet market platforms. It will then examine Embraer’s entrance into the Chinese market, where the company suffered from several missteps and eventually had to refocus its business model from commercial to executive jets. Finally, as globalization continues to “emancipate international business from its institutional and social constraints,” (Muldoon 2005) this paper will address how the relatively new and emerging discipline of Business Diplomacy is continuing to mature and grow in stature and influence through the proposition of a new challenge or “liability” that corporations must also overcome as they expand into new markets. Through the analysis of Embraer in China, this paper will introduce the Liability of Governance to the lexicon of Business Diplomacy and propose specific steps that a company can undertake to avoid it.