Made available in DSpace on 2015-04-14T13:37:24Z (GMT). No. of bitstreams: 1 398241.pdf: 671739 bytes, checksum: 6b2440575fac82dbcc9705c859adb1c1 (MD5) Previous issue date: 2008-01-09 / O presente estudo tem, por objetivos, analisar os pontos de aproxima??o entre a reportagem e a narrativa liter?ria e discutir a ficcionaliza??o da realidade pelo discurso jornal?stico. Para isso, o livro-reportagem Abusado: o dono do morro Dona Marta, do jornalista Caco Barcellos, ? examinado sob os pressupostos te?ricos do jornalismo e submetido ao m?todo de an?lise de enunciados ficcionais proposto por G?rard Genette, em que s?o contemplados a temporalidade da narrativa efeitos de ordem, dura??o e freq??ncia, o modo regula??o da informa??o atrav?s da dist?ncia e da perspectiva e a voz marcas deixadas no texto pelo narrador. Tamb?m s?o discutidos os conceitos de not?cia, reportagem, literariedade, ficcionalidade e modeliza??o, bem como as t?cnicas empregadas pelo new journalism.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-11-29T15:26:01Z No. of bitstreams: 1 TES_EDUARDO_ROUSTON_JUNIOR_COMPLETO.pdf: 1861060 bytes, checksum: 9292f812da53fc225992fdee400f89eb (MD5) / Made available in DSpace on 2016-11-29T15:26:01Z (GMT). No. of bitstreams: 1 TES_EDUARDO_ROUSTON_JUNIOR_COMPLETO.pdf: 1861060 bytes, checksum: 9292f812da53fc225992fdee400f89eb (MD5) Previous issue date: 2016-08-29 / This paper aims to examine, through the prism of the press and parliament, the political role of the Federalist Party in the context of the First Brazilian Republic. From this analysis two points jump in sight: first, to defend the strengthening of representative government, set within the framework of a parliamentary Republic and, secondly, the strengthening of the federal Union on the state units by delivering a political centralization regime for the Brazilian state. These two main aspects that stand in the federalist speech at the national level, were strongly influenced by a regional character problems, experienced by Rio Grande do Sul during the castilhista-borgista regime of authoritarian and conservative feature: with respect to the southern state, denial that the castilhista situationism was around the representative government and its replacement by a "dictatorship"; with regard to the Union, the desired subject of this authoritarian interests of Chief Rio Grande State. In this sense, we analyze, through political press and parliamentary speeches of federalist opposition, the relationship had major thematic policies raised by maragato party at the national level with the situation south of Rio Grande, occasioned by castilhista-borgista authoritarianism, which the Federalist Party, throughout its history, has always been energetic opponent. / Este trabalho tem como objetivo analisar, sob o prisma da imprensa e parlamentar, a atua??o pol?tica do Partido Federalista no contexto da Primeira Rep?blica Brasileira. Dessa an?lise dois pontos saltam ? vista: em primeiro lugar, a defesa do fortalecimento do governo representativo, definido dentro dos marcos de uma Rep?blica parlamentarista e, em segundo lugar, o fortalecimento da Uni?o federal sobre as unidades estaduais, delineando um regime de centraliza??o pol?tica para o estado brasileiro. Estes dois aspectos principais, que se colocam no discurso federalista no ?mbito nacional, estavam fortemente influenciados por uma problem?tica de car?ter regional, vivida pelo Rio Grande do Sul durante o regime castilhista-borgista, de fei??o autorit?ria e conservadora: com rela??o ao estado sulino, a nega??o que o situacionismo castilhista fazia em torno do governo representativo e a sua substitui??o por uma "ditadura"; com rela??o ? Uni?o, a pretendida sujei??o desta aos interesses autorit?rios do Chefe do Estado rio-grandense. Nesse sentido, analisamos, atrav?s da imprensa pol?tica e dos discursos parlamentares da oposi??o federalista, a rela??o que possu?am as principais tem?ticas pol?ticas levantadas pelo partido maragato no plano nacional com a situa??o sul-rio-grandense, ensejada pelo autoritarismo castilhista-borgista, do qual o Partido Federalista, ao longo de sua hist?ria, foi sempre en?rgico opositor.

Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle.

Les propriétés de symétrie d'un objet représenté sous la forme d'un modèle B-Rep CAO sont analysées localement et globalement à travers une approche de type diviser pour conquérir. La surface frontière de l'objet est décrite à partir de surfaces canoniques fréquemment utilisées dans les formes de composants mécaniques. La première phase de l'analyse consiste en la génération de faces et d'arêtes maximales indépendantes du processus de modélisation de l'objet mais préservant ses propriétés de symétrie. Ces faces et arêtes constituent des ensembles infinis de points traités globalement. La seconde phase est l'étape de division consistant en la création de plan et axes de symétrie de candidats pour les faces et arêtes maximales générées précédemment. Enfin, suit l'étape de propagation de ces plans et axes de symétrie représentant la phase de conquête et déterminant les propriétés de symétrie locales et globales de l'objet et caractérisant ses zones non-symétriques.

Les conduites primaires des centrales nucléaires REP (304L et 316L) sont protégées contre la corrosion par une couche d'oxyde. Ces conduites, qui forment une barrière entre le milieu primaire et l'extérieur, subissent des phénomènes liés à la corrosion sous contrainte (assisté ou non par irradiation) pouvant entrainer des dommages. L'objectif de cette étude est de comprendre les phénomènes régissant les stades initiaux de formation des couches d'oxydes sur ces alliages en tenant compte de l'orientation cristallographique des grains sous-jacents. En utilisant des techniques de caractérisation avancées comme GIXRD, spectroscopie Raman, XPS, MEB et MET (BF, HRTEM, Astar, EELS et HREELS), sur les toutes premières étapes de l'oxydation, une séquence d'oxydation a été proposée pour l'alliage 316L.

Pathogen-derived resistance (PDR) strategies, particularly those based on post-transcriptional gene silencing, have been used with great success for the generation of transgenic plants with resistance to RNA viruses. In contrast, a suitable strategy for transgenic resistance to ssDNA plant viruses, including those viruses belonging to the Geminiviridae, has remained elusive. Further, there is no convincing evidence that either post-transcriptional gene silencing, or pathogen-derived resistance in general, would be broadly applicable to ssDNA plant viruses. Researchers at QUT have been developing a novel resistance strategy against ssDNA viruses based on virus-activated expression of a stably integrated suicide gene. The strategy, based on InPAct (In Plant Activation) technology, relies on a &quotsplit" suicide gene cassette being arranged in such a way that expression of a lethal ribonuclease (barnase) is dependent on the virus-encoded replication-associated protein (Rep). Upon infection, Rep mediates the release of the construct resulting in the reconstitution of a transcribable and translatable episomal suicide gene expression cassette. The research for this PhD describes the development of an InPAct vector designed to confer resistance to Tomato leaf curl begomovirus (ToLCV), a major cause of disease in Solanaceous crops in the tropics and subtropics. ToLCV-based InPAct vectors were constructed based upon two ToLCV isolates from Australia and North Vietnam. Prior to the generation of InPAct cassettes, the entire ToLCV-[Au] and ToLCV-Vie intergenic regions (IRs) were embedded within the castorbean catalase intron of a β-glucuronidase expression vector to determine the effect of the IR upon transcript processing. Using transient reporter gene assays in tobacco NT-1 cells, it was demonstrated that the ToLCV IRs both contained cryptic intron splice sites which interfered with efficient transcript processing and GUS expression. A series of truncations to the IRs were subsequently made to identify the potential cryptic intron splice sites and/or interfering sequences in both the ToLCV-[Au] and ToLCV-Vie IRs. The final truncated IRs, which were used in the construction the InPAct cassettes, comprised approximately 100 bp and appeared to contain all the necessary cis-acting elements required for efficient rolling circle replication (RCR). Using histochemical GUS assays and Southern analyses, the InPAct cassettes were shown to be activated and replicated only in the presence of the cognate viral Rep. GUS expression levels were shown to be further enhanced in the presence of the ToLCV replication-enhancer protein (REn) and by the addition of the Tobacco yellow dwarf mastrevirus origin of second strand synthesis into the cassette. Under these conditions, Rep-activated GUS expression from the InPAct vectors was found to reach levels similar to that of the benchmark CaMV 35S promoter. Fifteen independent transgenic lines containing the ToLCV-[Au] and -Vie InPAct-GUS cassettes were generated by Agrobacterium-mediated transformation of tobacco leaf discs. Using agroinfiltration and histochemical assays, Rep-mediated activation of the InPAct cassettes and subsequent GUS expression was demonstrated in 11 out of the 15 lines tested; six of which showed expression levels equivalent to, or higher than, that obtained using a CaMV 35S promoter control. Evidence for activation of the integrated InPAct cassettes at the molecular level was provided by Southern analyses, with showed both linear and open circular forms of the replicating InPAct episome in genomic DNA extracted from infiltrated leaf tissue. Following the demonstration of Rep-activatable reporter gene expression and episomal replication of the ToLCV-based InPAct-GUS vectors using transient and stable tobacco transformation assays, new ToLCV-based InPAct vectors were designed to express the lethal RNase, barnase, in an attempt to generate virus resistant plants. Although transient assays in NT-1 cells demonstrated some &quotleaky" expression of barnase from the InPAct vectors, the level of barnase-mediated cell death from the InPAct vectors was found to be significantly increased in the presence of the cognate Rep and REn. Thirteen independently transformed tobacco lines containing the ToLCV-[Au] InPAct-barnase cassette were generated by Agrobacterium-mediated transformation of tobacco leaf discs. However, agroinfiltration of these plants with ToLCV Rep and REn failed to activate a barnase response. Subsequent molecular analyses on two transgenic lines revealed that both contained mutations in the barnase-coding gene in a region known to encode the active site. These mutations were presumed to result from the leaky barnase expression during initial stages of the Agrobacterium transformation which would favour the selection of barnase mutant InPAct plants. To overcome the problems associated with leaky expression of barnase, a barstar-expression cassette was included in the ToLCV-[Au] InPAct-barnase cassette. Transient assays in non-transgenic tobacco leaves demonstrated that the basal levels of barstar expressed from the modified InPAct vector were sufficient to negate the effects of leaky barnase expression. Importantly, however, the level of barnase expression in the presence of Rep and REn was shown to be sufficient to overcome the basal levels of barstar. Seventeen independently transformed lines were generated with the ToLCV-[Au] InPAct-barnase/barstar cassette, and analysis of one line revealed the presence of an uncorrupted barnase-coding region. Using transient agroinfiltration assays, seven of the transgenic lines showed varying levels of cognate Rep and REn-activated, barnase-induced cell death. Fifteen transgenic lines were challenged with ToLCV-[Au] by injection of recombinant Agrobacteria containing an infectious ToLCV clone. Unfortunately, all lines displayed typical ToLCV symptoms and tested positive for virus by PCR at 28 days post-inoculation. The inability of the InPAct cassette to confer resistance to ToLCV may have been due to one or a combination of factors, including (i) a delay in barnase-induced cell death, (ii) homology-dependent silencing of the integrated cassette, (iii) generally low-level, Rep-activated barnase expression or (iv) excessive virus load due to the artifical method of inoculation. This study details the first report of a ToLCV-based InPAct system for Rep-induced transgene expression in planta. Despite failing to generate ToLCV-resistant plants, the research findings will provide a solid foundation to develop a more effective InPAct vector and ultimately assist in the generation of transgenic plants with resistance to ToLCV and potentially other ssDNA plant viruses, particularly the begomoviruses.

Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV. The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed. An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed. The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication. Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed. Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids. The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-28Bitstream added on 2014-06-13T20:37:45Z : No. of bitstreams: 1 rodrigues_lmr_me_botfca.pdf: 618458 bytes, checksum: cb654c56905a6abeae0bdd7484f37739 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho teve como objetivo avaliar a agressividade de linhagens de Ralstonia solanacearum provenientes de solanáceas, plantas ornamentais e eucalipto, em plantas de batata, tomate e fumo, bem como caracterizar as linhagens por meio de técnicas moleculares. Vinte e duas linhagens foram utilizadas nos ensaios de avaliação da agressividade, em experimentos conduzidos em casa-de-vegetação evidenciaram alta severidade da doença pelas linhagens de R. solanacearum quando inoculadas em plantas de tomate e batata, sendo a batata mais afetada nas inoculações. Todas as linhagens mostraram-se agressivas, sendo que o fumo mostrou baixa suscetibilidade ao ataque das bactérias. As linhagens mais agressivas em plantas de tomate foram IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 e IBSBF 2000, pertencentes às biovares I, II e III. As linhagens mais agressivas às plantas de fumo foram IBSBF 309, IBSBF 2131 e IBSBF 292T, pertencentes à biovar I. Foi efetuado também ensaio de microbiolização in vitro em sementes de eucalipto, a fim de se identificar possíveis linhagens patogênicas a esta espécie vegetal e concluiu-se que todas as linhagens utilizadas infectaram plantas de eucalipto ou afetaram seu crescimento. A caracterização molecular de 41 linhagens de Ralstonia solanacearum, provenientes de diversas plantas hospedeiras, incluindo solanáceas, bananeira, helicônia, plantas ornamentais e eucalipto, foi efetuada empregando-se ERIC e BOX-PCR e os resultados mostraram grande diversidade genética entre as linhagens. A análise de PCR-RFLP da região espaçadora 16S-23S DNAr permitiu distinguir os isolados pertencentes à biovar III das demais biovares (I, II, IIA e IIT), quando digeridos com as enzimas Taq I e Hin6 I. A análise de sequenciamento de parte dos genes Endoglucanase (Egl) e MutS possibilitou a classificação em filotipos e os resultados... / This study aimed to evaluate the aggressiveness of strains of Ralstonia solanacearum from solanaceus, ornamental and eucalyptus plants, on potato, tomato and tobacco, and to characterize the strains through molecular techniques. Twenty-two strains were used in this study to evaluate the aggressiveness and, the experiments conducted in a greenhouse revealed the high susceptibility of tomato and potato plants, with the potato being the most affected on through the inoculations. All isolates proved to be aggressive and higher tolerance to the attack of bacteria was verified on tobacco plants. Strains more aggressive on tomato were IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 and IBSBF 2000, belonging to biovars I, II and III. The more aggressive strains on the tobacco plants were IBSBF 309, IBSBF 292T and IBSBF 2131 belonging to biovar I. Tests in vitro of microbiolization of eucalyptus seeds were also performed in order to identify possible pathogenic strains to this species and the results showed that all strains used cause infection on emerging plants or affected their growth. To molecular characterization of 41 strains of Ralstonia solanacearum from several host plants including solanaceous, banana, heliconia, ornamentals and eucalyptus were employed to ERIC and BOX-PCR, and the results showed high genetic diversity among strains. The analysis of PCR-RFLP of 16S-23S spacer region rDNA allowed us to distinguish the isolates belonging to biovar III from the others (biovars I, II, IIA and IIT) when digested with enzymes Taq I and Hin6 I. The sequence analysis of the partial of Endoglucanase (Egl) and MutS genes allowed the classification in phylotypes and the results revealed a predominance of the phylotype II in Brazil, and four isolates were classified in the phylotype I, all belonging to biovar III

Alternative energy technology has been used widely in rural electrification program (REP) all over the world for many years now. Renewable energy sources, such as solar, wind and biomass, are the preferred choices given the abundant resources available on site and the sophistication of the technologies involved. Combinations of two or more of the resources, together with an energy storage system and occasionally a conventional energy generator, create a hybrid system, which is reliable and durable. In Malaysia, solar photovoltaic (PV) base systems, implemented on a large scale, can provide round-the-clock electricity services for areas that are inaccessible by the electricity grid network. One of Malaysia s REP initiatives is solar PV-diesel hybrid systems for 160 schools in rural Sabah. The systems have been in operation for several years, but studies in the program are limited. Thus, understanding the system operation and functional is a highly valuable experience and lessons can be learned for implementation of the rural electrification program (REP). The overall aim of the research is to evaluate the REP in social, organizational, technical and economic aspects of the program that the findings can facilitate the stakeholders, such as the policy makers and implementers for current and future approaches, measures and decisions on REP activities and initiatives in Malaysia. This thesis has described the approaches on investigating the rural school s electrification program in Sabah. Analysis of system operation and function is conducted by examining and evaluating the recorded data from the system. A set of technical indicators is introduced in the form of system performance indicators and system reliability indicators. Furthermore, comparisons are made between the actual system operation and the optimum system configuration based on the actual data of the renewable energy resources, electricity energy consumption and costs in installation and operation. A field study was conducted at fifteen rural schools that use the solar PV system to determine the effectiveness of the program in transforming the rural schools to better learning environments and livelihoods. The findings indicate that most system components were found to be in good operation, and the operation of the solar PV system agreed to the indicators of system performance and system reliability. Additionally, the system reliability indicators can be seen as a vital tool not only to identify the values of the system capacity but also for prediction measures in analysing the durability of each component. The analysis of the actual system operation provides optimum values in terms of technical indicators, whereas the optimized system shows economic advantages. The findings show a high degree of responses from the end users in the level of satisfaction, appreciation, motivation and academic excellence. Nevertheless, several improvements are required to enhance the sustainability elements of the REP, especially from the organizational and governance perspectives. These includes effective coordination among the rural development-related agencies, the improvement on the transition between installation and maintenance work, efficient reporting process and training and awareness program need to be extended to every end user for sustainability in information and knowledge.