Dissertations / Theses on the topic 'CTV'

Citrus tristeza virus (CTV) is present in almost all of the major citrus production areas where it continues to reduce the profitability of citriculture worldwide. Severe stem-pitting strains are endemic to Southern Africa. In addition to making use of decline-tolerant rootstocks, the implementation of a mild-strain cross-protection program has sought to increase the citrus production in the region. Of all the commercially cultivated citrus species, grapefruit cultivars are among the most susceptible to the severe stem-pitting strains and infections often lead to a breakdown of cross-protection. Recent research has shown that CTV crossprotection operates through a strain-specific mechanism, which relies on a virusspecific protein, expressed from the p33 gene. The specificity of this mechanism has highlighted the need for determining the CTV diversity within production areas as well as accurately characterising CTV cross-protection sources that will be capable of preventing secondary inoculations of severe strains in the field. The accurate characterisation of CTV populations, which are usually made up of a number of disparate strains, requires the use of robust PCR protocols. Mismatches between primers and their corresponding binding sites may introduce primer-associated bias during amplification. The primer-associated bias of four sets of CTV specific primers, targeting the A and F regions and the p23 and p33 genes, were evaluated. This was done through the amplification of defined templates followed by their characterisation using the sequencing of multiple clones, as well as Illumina next generation sequencing. High levels of bias were found to be associated with the primer pairs targeting the A and F regions. The p33 gene primers were found to be biased against two genotypes and suggestions for preventing this apparent bias are discussed. The primer pair targeting the conserved p23 gene was found to have very little associated bias. The second major aspect of this study was the large scale survey of CTV diversity of pre-immunised Star Ruby grapefruit orchards in Southern Africa. Samples were collected from eight Star Ruby production sites throughout Southern Africa, namely Hoedspruit 1, Hoedspruit 2, Malelane 1, Malelane 2, Swaziland (Mananga), Northern Cape (Kakamas), Sundays River Valley and Nkwalini Valley, where between 16 and 32 samples per site were collected. The p33 gene was amplified for each of the collected samples and subjected to direct Sanger sequencing. A protocol making use of Illumina MiSeq sequencing was established and used to sequence 96 samples. A subset of six samples was selected for cloning, which resulted in a total of 218 sequenced clones and compared with that of the Illumina sequencing data. High levels of CTV diversity were observed between orchards, as well as between different trees within the same orchard. Most of the populations were made up of a single dominant group, sometimes with several minor sequence types. Sequence reads corresponding to strains within the Resistance Breaking (RB) genotype were most numerous, especially in the most recently planted orchards and were present within all of the populations analysed. The Kpg3/SP/T3 group appeared to be represented the second most prevalent genotype and seems to become more common as the orchard ages. Genotypes of the HA 16- 5, VT, AT-1, T36, Taiwan-Pum/M/T5 and T30 types, were represented sporadically and at variable levels in populations from numerous collection sites. This has been one of the most extensive diversity studies of CTV to date and has provided unprecedented baseline knowledge of CTV diversity in Southern Africa.
Thesis (PhD)--University of Pretoria, 2015.
Microbiology and Plant Pathology
PhD
Unrestricted

O presente trabalho é uma investigação sobre o processo de profissionalização da defesa em direitos humanos no Brasil, considerado a partir do estudo de caso da Comissão Teotônio Vilela (CTV). Fundada em 1983, em meio à transição democrática, a CTV inicialmente trabalhou com a defesa dos direitos de presos comuns e, conforme o processo de consolidação democrática avançava, sua agenda de reivindicações foi alterada. Paralelamente, a trajetória dos seus membros também é analisada a fim de mostrar como eles se trabalharam individualmente para a promoção e proteção dos direitos humanos no Brasil. A pesquisa foi realizada por meio de entrevista com os membros fundadores da CTV, bem como nos arquivos da entidade sediada no Núcleo de Estudos da Violência (NEV/USP), levando-se em conta os seus relatórios, relatos de visitas, recortes de jornais e processos elaborados a fim de buscar alguma reivindicação. Os resultados sugerem que a defesa dos direitos humanos no Brasil foi perpassada por dois aspectos, a saber: o aumento da participação no Estado e em governos e também no processo de internacionalização das reivindicações.
The present work is an investigation about the process of professionalization of human rights defense in Brazil, considered through the case study of Comissão Teotônio Vilela (CTV). Founded in 1983, during the democratic transition, CTV started its work with the defense of regular prisoners and, with the democratic consolidation advancement, its claim agenda was either changed. In parallel, the trajectory of its members is also analyzed to show how they worked individually for the promotion and protection of human rights in Brazil. The research was conducted through interviews with founding members of the CTV, as well as the archives of the entity based at Center for the Study of Violence (NEV / USP), taking into account their reports, reports of visits, newspaper clippings and processes designed to seek some claim. The results suggest that the defense of human rights in Brazil was pervaded by two aspects, namely: increasing participation in state and governments and also in the internationalization process of claims.

O vírus da tristeza dos citros (CTV) é uma das maiores ameaças à citricultura mundial. No Brasil, mesmo com a pré-imunização e com a substituição de porta-enxertos, estirpes fortes de CTV ainda causam prejuízos consideráveis. Com o aparecimento da Morte Súbita dos Citros em 1999 e a possível relação desta doença com o CTV, este vírus voltou a figurar como patógeno de importância no cenário da citricultura brasileira. Uma das possíveis soluções para o controle de viroses em fruteiras é a obtenção de plantas transgênicas resistentes ou imunes. O objetivo deste trabalho foi avaliar a resistência ao CTV de plantas transgênicas de laranja \'Hamlin\' contendo três construções gênicas oriundas de seqüências do genoma do CTV. Estas construções gênicas visaram ativar rotas de RNAi (hairpin da capa protéica e seqüência conservada antisenso do CTV) e mecanismos de defesa relacionados à expressão da capa protéica do CTV. As plantas transgênicas foram desafiadas com uma estirpe fraca de CTV (CTV-IAC) por meio de borbulhas e pulgões pretos (Toxoptera citricida Kirkaldy) contendo o vírus. A avaliação da resistênica à replicação viral foi feita por análises de ELISA. As plantas transgênicas foram consideradas não resistentes à infecção e translocação viral quando inoculadas com borbulhas. Entretanto algumas plantas mostraram retardamento da infecção. Não foi possível determinar se houve resistência à transmissão de CTV por pulgões já que a técnica utilizada não foi capaz de infectar os controles de maneira uniforme.
The Citrus tristeza virus (CTV) is one of the greatest threats to the citrus industry worldwide. In Brazil, CTV continues to cause damage through strong strains despite the use of techniques like cross-protection and substitution of intolerant rootstocks. With the appearance and spread of the Citrus Sudden Death disease in 1999 and its possible relation to CTV, this virus was again among important pathogens within the Brazilian citrus industry. One of the possible solutions for controlling virus diseases in fruit crops is the development of immune or resistant transgenic plants. The objective of this work was to evaluate the resistance to CTV of transgenic \'Hamlin\' sweet orange plants containing three transgenic constructs obtained from CTV genomic sequences. The genetic constructs used aimed to activate RNAi defense routes (coat protein hairpin and a conserved sequence from CTV) and resistance mechanisms related to the coat protein expression. The transgenic plants were challenged with a weak strain of CTV, CTV-IAC, by bud and aphid (Toxoptera citricida Kirkaldy) inoculation. The evaluation of viral replication was done by ELISA analysis. The transgenic plants were considered susceptible to viral replication and translocation when bud inoculated. However, a few plants showed retardation of infection. It was not possible to determine resistance in the aphid transmission assay since the controls were not uniformly inoculated.

The purpose of this study was to estimate the CTV-PTV margin required for prostate and head and neck cancer treatments at the radiotherapy departments of Karolinska University Hospital.    Portal image data from patients treated at the radiotherapy departments during the period of 2009-2011 was used to estimate the set-up displacements for each treatment area. By using the acquired images the magnitude of the systematic, i.e. preparatory, and random, i.e. execution, error was determined in the anterior-posterior (AP), superior-inferior (SI) and right-left (RL) direction. The calculated PTV margin is based on the systematic and random errors of the entire patient populations. A total of 40 patients were used for the analysis of prostate treatments and 47 patients for head and neck treatments. The evaluation of the PTV margin was done for three different matching protocols; no matching (skin marker alignment), five day matching and daily matching.      With no image verification in prostate treatments the calculated PTV margin taking both inter- and intrafractional errors into account was 13.6, 9.2 and 7.9 mm in AP, SI, and RL direction respectively. The corresponding PTV margin in head and neck treatments was found to be 6.7, 5.3 and 4.9 mm. Using a five day matching protocol of the bony anatomy showed no considerable reductions in margins for neither prostate nor head and neck treatments. With daily matching of the bony anatomy in prostate treatments the calculated margins was reduced to 8.1, 7.9 and 2.4 mm in the AP, SI and RL direction respectively.  Measurements of the residual deviations of individual cervical vertebrae after daily image verification and correction in head and neck cancer treatments showed that all matching protocols will require larger margins in the lower vertebrae in order to account for the set-up error in the AP direction. The corresponding margins needed using daily matching of the bony anatomy would be 3.9, 5.4 and 6.0 mm for C1, C4 and C5 respectively in the AP direction.    In the absence of daily imaging the currently used PTV margins might be inadequate for covering to movement of the targets. The deviations in the AP direction of the cervical vertebrae in head and neck cancer treatments should be investigated further in order to ensure that the motion of the target is covered and that no risk organs are subjected to harmful dose levels.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Devido a grande importância econômica da citricultura no Brasil e mundo e aos problemas sanitários enfrentados sendo alguns limitantes para o cultivo, como é o caso das doenças causadas por vírus como: a tristeza cítrica, causada pelo vírus da tristeza dos citros (CTV), pertencente ao gênero Closterovirus, família Closteroviridae, uma das maiores ameaças da citricultura mundial, mesmo com a pré imunização através de estirpes fracas do vírus e substituição de porta enxertos, estirpes fortes de CTV ainda causam prejuízos consideráveis. E com o aparecimento da doença morte súbita dos citros (MSC) de etiologia não determinada. Pelo fato de não haver ainda métodos eficientes de separação de ambos os vírus presentes em uma única amostra, levantando se as hipóteses que a causa da MSC esteja relacionada a uma estirpe do vírus CTV, a um vírus do gênero Marafivirus denominado Citrus Sudden Death-associated Virus (CSDaV), pertencente ao gênero Marafivirus, família Tymoviridae, ou a uma associação entre eles. Este trabalho vem propor um método eficaz de separação por citometria de fluxo (FC) de CTV e CSDaV em amostras semi purificadas, diluídas em tampão TE, pH7,5, utilizando marcação de ácidos nucléicos com Iodeto de Propídeo (PI) e conjugação de anticorpos policlonais anti CTV com Isotiocianato de Fluoresceína (FITC), cuja eficácia do método foi comprovada pela reação da polimerase em cadeia (PCR)
Because of high economic importance of citrus in Brazil and the world and health problems being faced some limiting factors for growing as is the case of diseases caused by viruses such as sadness citrus caused by citrus tristeza virus (CTV) belonging to Closterovirus gender, family Closteroviridae, one of the biggest threats to the citrus industry worldwide, even with the pre immunization using mild virus strains and replacement of the rootstocks, strong strains of CTV still cause considerable damage. And with the onset of the disease citrus sudden death (MSC) of undetermined etiology. Because there is not yet efficient methods of separation of the two viruses present in a single sample, raising the hypotheses that the cause of SCD is related to a strain of CTV, a virus Marafivirus group called Citrus Sudden Death-associated Virus (CSDaV) belonging to the genus Marafivirus, Tymoviridae family, or an association between them, this paper proposes an effective method of separation by flow cytometry (FC) and CTV in samples CSDaV semi purified, diluted in TE buffer, pH7, 5, using marking of nucleic acids with propidium iodide (PI) and a combination of polyclonal anti CTV with Fluorescein isothiocyanate (FITC), the effectiveness of the method was confirmed by polymerase reaction chain reaction (PCR)

Orientador: Marcos Antonio Machado
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Estudos preliminares sobre a localização de epítopos na proteína do capsídeo (CP) de três isolados de CTV ('Pêra IAC', 'Pêra CB-22' e 'Pêra CB-104') foram realizados em três etapas. Na primeira, foi realizada a amplificação de oito regiões distintas do gene da CP, codificantes para três regiões Nterminais (aa 1 até 70; aa 1 até 120; aa 1 até 170), três regiões C-terminais (aa 70 até 223; aa 120 até223; aa 170 até 223) e duas regiões internas da CP (aa 37 até 67 e aa 64 até 94) onde haviam sido determinadas diferenças significativas entre as CPs dos três isolados. Na segunda etapa, foram avaliadas duas estratégias para expressão dos peptídeos de interesse: clonagem em vetor de expressão através de ligação AT (vetor Pinpoint TM Xa-1 T, Promega) e clonagem das seqüências alvo em vetores do sistema de expressão pET. O vetor Pinpoint TM Xa-1 T, carreando como inserto o gene inteiro da CPCTV, obtido como produto de PCR após amplificação do cDNA dos três isolados, transformou células competentes de E. coli JM109, mas após indução com IPTG, não houve expressão da proteína de fusão esperada. Os vetores do sistema pET, após ligação das seqüências alvo, não transformaram células competentes de E. coli BL21(DE3)pLysS. Na terceira etapa, três proteínas recombinantes (MBP-CPCTV, CB-22 e CB-104) produzidas a partir da expressão do gene da CP-CTV dos três isolados, foram clivadas com brometo de cianogênio. Os produtos de clivagem foram avaliados através de '¿Western Blotting¿ contra anticorpos monoclonais específicos para CTV. O monoclonal IC-04.6, desenvolvido contra a proteína recombinante CB-22 detectou epítopos, numa reação intensa, em peptídeos com massa estimada em 27 kDa e 18 kDa, originários da CB-22; o monoclonal 39-08, desenvolvido contra a proteína recombinante CB-104 detectou epítopos, numa reação moderada, em peptídeos com massa estimada de 28 kDa e 14 kDa, originários qa CB-104; o monoclonal MCA-13, desenvolvido contra um isolado da Florida, detectou epítopos em peptídeos originários das proteínas recombinantes MBP-CPCTV e CB-104; o monoclonal 3DF1, desenvolvido contra isolados de CTV espanhóis, detectou epítopos em peptídeos originários da CB-22 e CB-104 e o monoclonal 3CA5, desenvolvido contra isolados espanhóis de CTV, detectou epítopos apenas num peptídeo com massa estimada de 18 kDa presente na CB-22. Estes resultados sugerem que diferentes epítopos são reconhecidos pelos cinco monoclonais avaliados e que serão necessárias novas estratégias para avaliá-los
Abstract: Preliminary studies about the epitopes location in the capside protein of the isolates of the citrus tristeza virus of three isolate ('Pêra IAC', 'Pêra CB-22' and 'Pêra CB-104') were accomplished. In the first phase, the amplification was accomplished with specific direct and reverse primers of eight different regions of the CP gene, coding for three N-terminal peptides (aa 1 to 70, aa 1 to 120 and aa 1 to 170), three C-terminal peptides (aa 70 to 223, aa 120 to 223 and aa 170 to 223) and two internal peptides of CP (aa 37 to 64 and aa 64 to 90). In the second phase they were appraised two strategies for expression of the peptides: cloning in expression vector through AT cloning site (vector Pinpoint TM Xa-1 T promega), and cloning of the coding sequences in vectors of the system pET. Vector Pinpoint TM Xa-1, containing as insert the whole gene of CP, obtained as PCR's product after amplification of the three isolates' cDNA, transformed competent cells E. coli JM109, but after induction with IPTG there was not expression of the peptides of interest. The vectors of the pET system didn't transform competent cells E. coli BL21 (DE3)pLysS. In the third phase, three recombinant proteins (MBP-CPC1V, CB-22 and CB-104), produced from the expression of the CP-C1V gene of the three isolates, they were broken with cyanogen bromide. The break products were evaluated through "Western Blotting" against monoclonal antibodies specific for CTV. The monoclonal IC-04.6, developed against the recombinant protein CB-22 detected epitopes, in an intense reaction, in peptides with mass esteemed in 27 kDa and 18 kDa, original of CB-22; the monoclonal 39-08, developed against the recombinant protein CB-104 detected epitopes, in a moderate reaction, in peptides with esteemed mass of 28 kDa and 14 kDa, original of CB-104; the monoclonal MCA-13, developed against the T-36 isolate from Florida, detected epitopes in original peptides of the recombinants proteins MBP-CPCTV and CB-104; the monoclonal 3DF1, developed against Spanish isolates of CTV, detected epitopes in original peptides of CB-22 and CB-104 and the monoclonal 3CA5, developed against Spanish isolates of C1V, just detected epitopes in a peptide with esteemed mass of 18 kDa present in CB-22. These results suggest that the five monoclonal recognizes different epitopes and that will be necessary new strategies to evaluate them
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

Orientador: Paulo Inácio da Costa
Banca: Henrique Ferreira
Banca: Nelson Wulff
Banca: José Belasque Junior
Banca: Marcel Bellato Spósito
Resumo: Devido a grande importância econômica da citricultura no Brasil e mundo e aos problemas sanitários enfrentados sendo alguns limitantes para o cultivo, como é o caso das doenças causadas por vírus como: a tristeza cítrica, causada pelo vírus da tristeza dos citros (CTV), pertencente ao gênero Closterovirus, família Closteroviridae, uma das maiores ameaças da citricultura mundial, mesmo com a pré imunização através de estirpes fracas do vírus e substituição de porta enxertos, estirpes fortes de CTV ainda causam prejuízos consideráveis. E com o aparecimento da doença morte súbita dos citros (MSC) de etiologia não determinada. Pelo fato de não haver ainda métodos eficientes de separação de ambos os vírus presentes em uma única amostra, levantando se as hipóteses que a causa da MSC esteja relacionada a uma estirpe do vírus CTV, a um vírus do gênero Marafivirus denominado Citrus Sudden Death-associated Virus (CSDaV), pertencente ao gênero Marafivirus, família Tymoviridae, ou a uma associação entre eles. Este trabalho vem propor um método eficaz de separação por citometria de fluxo (FC) de CTV e CSDaV em amostras semi purificadas, diluídas em tampão TE, pH7,5, utilizando marcação de ácidos nucléicos com Iodeto de Propídeo (PI) e conjugação de anticorpos policlonais anti CTV com Isotiocianato de Fluoresceína (FITC), cuja eficácia do método foi comprovada pela reação da polimerase em cadeia (PCR)
Abstract: Because of high economic importance of citrus in Brazil and the world and health problems being faced some limiting factors for growing as is the case of diseases caused by viruses such as sadness citrus caused by citrus tristeza virus (CTV) belonging to Closterovirus gender, family Closteroviridae, one of the biggest threats to the citrus industry worldwide, even with the pre immunization using mild virus strains and replacement of the rootstocks, strong strains of CTV still cause considerable damage. And with the onset of the disease citrus sudden death (MSC) of undetermined etiology. Because there is not yet efficient methods of separation of the two viruses present in a single sample, raising the hypotheses that the cause of SCD is related to a strain of CTV, a virus Marafivirus group called Citrus Sudden Death-associated Virus (CSDaV) belonging to the genus Marafivirus, Tymoviridae family, or an association between them, this paper proposes an effective method of separation by flow cytometry (FC) and CTV in samples CSDaV semi purified, diluted in TE buffer, pH7, 5, using marking of nucleic acids with propidium iodide (PI) and a combination of polyclonal anti CTV with Fluorescein isothiocyanate (FITC), the effectiveness of the method was confirmed by polymerase reaction chain reaction (PCR)
Doutor

Los aislados de CTV están formados generalmente por una mezcla de variantes de secuencia, que se generan mediante procesos de mutación y recombinación. Su frecuencia en la población es el resultado de distintas presiones selectivas y de fenómenos de migración y deriva genética, generalmente asociados a los procesos de transmisión. El objetivo de esta tesis era analizar por separado el efecto de diversos factores de los procesos de transmisión sobre la estructura poblacional y la diversidad genética de distintos aislados del virus. Se analizó el efecto de la transmisión por pulgón. Ésta ocurrió con baja eficiencia y en general, las secuencias de los genes analizados en las plantas receptoras fueron idénticas a las de las plantas donantes y el aislado de colección. La transmisión por injerto y/o pulgón supone un cuello de botella que puede dar lugar a un efecto fundador. Se analizó el efecto de la transmisión por injerto entre plantas de una misma especie huésped y no se observaron cambios llamativos en la estructura y diversidad poblacional. También se estudió si la transmisión por injerto de dos aislados de CTV a diferentes especies y variedades comerciales propagadas sobre citrange Carrizo podía inducir cambios en la población. Aunque no observamos cambios significativos en la diversidad genética, la estructura poblacional se vio alterada en algunas plantas. Esta alteración fue debida a la aparición de nuevas variantes de secuencia cercanas filogenéticamente que llegaban a predominar en dichas poblaciones. Finalmente, se utilizó un aislado clonal de CTV para el estudio de la variación genética de novo en distintos huéspedes a lo largo del tiempo. Se efectuaron pases sucesivos por injerto de dicho aislado desde su huésped original a distintos huéspedes susceptibles, y a un huésped parcialmente resistente (naranjo amargo). Las poblaciones en los huéspedes susceptibles presentaron una elevada estabilidad, pero los pases sucesivos en el huésped naranjo amargo dieron lugar a cambios importantes en la población, debido a la aparición de variantes de secuencias muy divergentes anteriormente halladas en dos aislados naturales del virus. La detección de secuencias minoritarias cuya posición filogenética se sitúa entre los tres genotipos avala la idea de una evolución entre el genotipo original y los otros dos detectados para intentar adaptarse al huésped. El hallazgo de secuencias recombinantes entre los tres genotipos apoya la presencia de éstos en el huésped naranjo amargo. La inoculación de huéspedes susceptibles a partir de plantas de naranjo amargo puso de manifiesto la progresiva pérdida de infectividad de la población mantenida en esta especie, debido en parte a la baja acumulación de CTV en la misma. Además fue imposible detectar el virus en brotes de lima propagados sobre estas plantas, lo que sugiere que además del bajo título viral algún factor del huésped pudiese dificultar el paso de CTV desde naranjo amargo a lima.
CTV isolates are composed of a population of sequence variants, resulting from mutation and recombination events. The frequency of these variants in the population is the outcome of different selective pressures, and migration and genetic drift phenomena generally associated to transmission processes. Here we analyzed separately the effect of different factors of the transmission process on the diversity and population structure of CTV isolates. We studied the effect of aphid transmission by nucleotide sequence comparisons between the donor and receptor plants and the collection CTV isolate. Transmission efficiency was low, and no sequence variation was observed. Aphid and graft transmission are a bottleneck resulting in a founder effect. We analysed the effect of graft transmission to a host of the same species. No obvious changes were observed in the CTV population structure and diversity We also studied if graft-transmission to different varieties propagated on Carrizo citrange could alter the CTV population. Although significant diversity changes were not observed, the population structure was occasionally altered due to the appearance of new sequence variants genetically close that became predominant in these populations. Finally, we used a clonal CTV isolate to study genetic variation generated de novo in different hosts. This isolate was serially passed by graft-transmission in different susceptible hosts and in a partially resistant host (sour orange). While CTV population was stable in the susceptible hosts, passages through sour orange caused major changes due to the appearance of new diverged sequence variants previously found in two natural isolates). Detection of minor variants phylogenetically located between these three genotypes supports the idea of an evolutionary process between the original and two new genotypes to get adapted to sour orange. Recombination events involving the three genotypes supports the presence of more than one genotype in infected sour orange. Inoculation from sour orange to susceptible hosts showed progressive loss of infectivity, due in part to the low virus titer in this host. CTV could neither be detected in Mexican lime propagated on these sour orange plants, suggesting that some host factor might also block CTV movement from sour orange to lime.

Citrus tristeza virus (CTV) is a viral species of the Closterovirus genus, having a ¿20 kb single-stranded and positive sense genomic RNA (gRNA) organized into 12 open reading frames (ORFs) potentially coding 17 protein products, some of which of unknown function. CTV proteins p25 and p27 belong to a gene block involved in virion assembly. It has been demonstrated that p25, along with p20 and p23, are RNA silencing suppressors in some species of Nicotiana, being the latter a pathogenesis determinant. CTV host range is very restricted and in nature viral infections are limited to the phloem cells of some citrus species. Consequently, working with this virus-host pathosystem requires an appropriate experimental host and an efficient CTV genetic system. In our laboratory we have developed a new CTV genetic system based on the agroinfection of Nicotiana benthamiana plants, a non-natural host, with infectious clones of the T36 isolate. That infection induces characteristic symptoms, some of which correlate with those caused by the virus in citrus plants. CTV-N. benthamiana interactions are very variable and genotype-dependent, so only some isolates can replicate in this species and T36 is unique causing systemic infections. In this work, we studied the function of CTV p20 and p25 proteins from three different isolates differing in its pathogenic characteristics. Transient expression of p20 and p25 fused to fluorescent proteins showed an identical subcellular localization for both N. benthamiana and citrus. The p20 protein of T36, T318A and T385 isolates localized in cytoplasm and nucleus forming amorphous aggregates associated with perinuclear regions, together with nuclear punctuate inclusions. On the other hand, subcellular localization of p25 differed depending on the CTV isolate. While p25 of T36 and T385 localized in the nucleus, that of T318A did it in cytoplasm. A detailed analysis of the protein regions involved in this subcellular localization unveiled a leucine-rich nuclear export signal (NES) in the N-terminus (Nt) of the protein. The pathogenic ability of CTV p20 and p25 in N. benthamiana was tested through a PVX heterologous viral infection, showing that p25 was not a pathogenicity determinant in this species, albeit p20 it was. Besides, the interactome of p25-T36 was more complex and diverse than that of p25-T318A, involving a greater number of metabolic processes, redox activities, homeostasis and cellular transport, biosynthesis and protein degradation, plastid and nucleic acid protein binding, biotic and oxidative stress, jasmonic-mediated defense, methylation, ROS signalling and HSP proteins. On the other hand, most p25-T318A potential interactors were related to transport/localization and stress response, like apoptosis, pathogenesis-related and HSP proteins, Ca+2-binding proteins and redoxins. Moreover, the experimental evolution of CTV in N. bethamiana was achieved through serial passages. The evolution process showed adaptative traits as the increase of graft survival, infectivity rates and viral titers, and a decreased time for the onset of symptom development in this species. Adaptive characteristics of evolved viral populations in N. bethamiana, were also correlated to their genetic variability and population structure. Two different evolved lineages showed convergent evolution processes reflected also at the molecular level. CTV viruses evolved in N. benthamiana were found to be less infectious at initial stages of infection when they were back-inoculated to citrus plants, and showed a decreased viral titer during the first year post-inoculation. This re-adaptation of N. benthamiana viral populations back to citrus was correlated, at a molecular level, with the progressive loss of the mutations appeared during its evolution process in N. benthamiana.
El virus de la tristeza de los cítricos (CTV) es un closterovirus con un RNA genómico (gRNA) de ssRNA (+) y ¿20 Kb organizado en 12 pautas de lectura abierta (ORFs) que codifican al menos 17 proteínas, algunas de función desconocida. Las proteínas p25 y p27 forman parte de un grupo de genes implicados en el ensamblaje del virión, y se ha demostrado que p25 junto con p20 y p23 son supresores de silenciamiento génico en algunas especies de Nicotiana, y la última es también un determinante de patogénesis. CTV tiene una gama de huéspedes muy restringida y de forma natural sólo infecta el floema de algunas especies de cítricos. Por ello, para trabajar con este patosistema virus-huésped, se requiere un huésped experimental adecuado y un sistema genético eficiente de CTV. Durante los últimos años hemos desarrollado un sistema genético de CTV basado en la agroinfección sistémica de Nicotiana benthamiana, una especie no-natural de este virus, con clones infecciosos del genotipo T36. Dicha infección va acompañada de síntomas característicos, algunos de los cuales son similares a los que el virus induce en cítricos. La interacción CTV-N. benthamiana es muy variable y genotipo-dependiente, sólo algunos aislados se replican en esta especie y únicamente T36 la infecta sistémicamente. En este trabajo, abordamos la función de las proteínas p20 y p25 de tres aislados de CTV que difieren en sus características patogénicas. La expresión transitoria de las p20 y p25 fusionadas a proteínas fluorescentes reveló su idéntica localización subcelular en N. benthamiana y cítricos. La proteína p20 se localizó en el citosol y el núcleo celular en agregados amorfos asociados a regiones perinucleares e inclusiones nucleares puntuales. En cambio, la expresión de la p25 de T36, T318A y T385 de CTV reveló una localización diferencial. Mientras la de T36 y T385 fue nuclear, la de T318A fue citosólica. Un análisis detallado de las regiones implicadas en dicha localización, reveló la existencia de una señal de exportación nuclear NES rica en leucinas en la región Nt de la proteína. Un análisis de la capacidad patogénica de p20 y p25 en N. benthamiana en un contexto de infección viral heterólogo a través de PVX, mostró que p25 no es un determinante de patogenicidad en esta especie, pero p20 sí. Por otra parte, el interactoma de la p25-T36 resultó mucho más complejo y diverso que el de la p25-T318A, interviniendo potencialmente en mayor número de procesos metabólicos de fotosíntesis, actividad redox, homeóstasis y transporte celular, biosíntesis y degradación de proteínas, unión a proteínas de los plastidios y ácidos nucleicos o de respuesta a estrés (biótico y oxidativo) o defensa mediada por la ruta del jasmónico, del ciclo de metilación, señalización por ROS y proteínas HSP. Las interacciones mayoritarias de la p25-T318A se relacionaron con transporte/localización y respuesta a estrés, principalmente con interactores implicados en procesos de apoptosis, patogénenis y proteínas HSP, de unión a calcio o redoxinas. También hemos conseguido la evolución experimental de CTV por pases seriados en N. benthamiana. Dicha evolución conlleva un conjunto de características adaptativas significativas como: el aumento del prendimiento de injertos, de la tasa neta de infectividad, del título viral y del adelanto de los síntomas causados en esta especie herbácea con el aumento de los pases. Las características adaptativas también se reflejaron a nivel molecular en la variabilidad genética y estructura de las poblaciones de los virus evolucionados en dos linajes independientes. Virus evolucionados en N. benthamiana resultaron menos infecciosos inicialmente por inoculación mecánica de regreso a cítricos, y se acumularon menos que el virus parental durante el primer año. Dicha re-adaptación de los virus evolucionados se reflejó a nivel molecular en la pérdida progresiva de las m
El virus de la tristesa dels cítrics (CTV) és un closterovirus amb un RNA genòmic de ssRNA(+) i 20 Kb organitzat en 12 pautes de lectura oberta (ORFs) que codifiquen, al menys, 17 proteïnes, algunes de les quals de funció desconeguda. Les proteïnes p25 i p27 formen part d'un grup de gens implicat en l'assemblatge del virió, i s'ha demostrat que p25, junt a p20 i p23, són supressors de silenciament gènic en algunes espècies de Nicotiana, i la última també és un determinant de patogènesi. La gama d'hostes de CTV és molt restringida i de forma natural sols infecta el floema d'algunes espècies de cítrics. Per això, per a treballar amb aquest patosistema virus-hoste, es requereix un hoste experimental adequat i un sistema genètic eficient de CTV. Durant els últims anys hem desenvolupat un sistema genètic de CTV basat en l'agroinfecció sistèmica de Nicotiana benthamiana, una espècie no-natural d'aquest virus, amb clons infecciosos del genotip T36. Aquesta infecció va acompanyada de símptomes característics, alguns dels quals són similars als que el virus indueix en cítrics. La interacció CTV-N. benthamiana és molt variable i genotip depenent, ja que sols alguns aïllats es repliquen en aquesta espècie i únicament T36 la infecta sistèmicament. En aquest treball hem abordat la funció de les proteïnes p20 i p25 de tres aïllats de CTV que difereixen en les seues característiques patogèniques. L'expressió transitòria de les p20 i p25 fusionades a proteïnes fluorescents va revelar la seua idèntica localització subcel·lular en N. benthamiana i cítrics. La proteïna p20 dels aïllats T36, T318A i T385 es va localitzar al citosol i al nucli cel·lular formant agregats amorfs associats a regions perinuclears, i inclusions nuclears puntuals. En canvi, l'expressió de la p25 de T36, T318A i T385 de CTV va revelar una localització diferencial. Mentre la de T36 i T385 fou nuclear, la de T318A fou citosòlica. Una anàlisi detallada de les regions implicades en eixa localització va revelar l'existència d'una senyal d'exportació nuclear (NES) rica en leucines a la regió Nt de la proteïna. L'anàlisi de la capacitat patogènica de p20 i p25 en N. benthamiana en un context d'infecció viral heteròloga a través de PVX, va mostrar que p25 no és un determinant de patogenicitat en aquesta espècie, però p20 sí. D'altra banda, l'interactoma de p25-T36 va resultar molt més complex i divers que el de p25-T318A, intervenint potencialment en un major nombre de processos metabòlics de fotosíntesi, activitat redox, homeòstasi i transport cel·lular, biosíntesi i degradació de proteïnes, unió a proteïnes dels plastidis i àcids nucleics o de resposta a estrés (biòtic i oxidatiu) o defensa mediada per la ruta del jasmònic, del cicle de metilació, senyalització per ROS i proteïnes HSP. Les interaccions majoritàries de la p25-T318A es relacionaren amb transport/localització i resposta a estrés, principalment amb interactors implicats en processos d'apoptosi, patogènesi i proteïnes HSP, d'unió a calci o redoxines. També hem aconseguit l'evolució experimental de CTV per passes seriats en N. benthamiana. Aquesta evolució comporta un conjunt de característiques adaptatives significatives com: l'augment de la supervivència dels empelts, de la taxa neta d'infectivitat, de la càrrega viral i de l'ajornament dels símptomes causats en aquesta espècie herbàcia amb l'augment dels passes. Les característiques adaptatives també es reflectiren a nivell molecular amb la variabilitat genètica i estructura de les poblacions dels virus evolucionats en dos llinatges independents. Virus evolucionats a N. benthamiana resultaren menys infecciosos inicialment per inoculació mecànica de tornada a cítrics, i s'acumularen menys que el virus parental durant el primer any. Aquesta re-adaptació dels virus evolucionats a N. benthamiana de tornada a cítrics es va reflectir a nivell molecular amb
Navarro López, JA. (2017). Estudio preliminar de las interacciones del virus de la tristeza de los cítricos (CTV) y su huésped [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90468
TESIS

La tristeza es la enfermedad viral más grave del cultivo de los cítricos y su agente causal es el virus de la tristeza de los cí­tricos (CTV). CTV induce uno o más de los siguientes sí­ndromes: I) decaimiento y muerte de los naranjos dulces (ND), pomelos y mandarinos injertados sobre el patrón naranjo amargo (NA), sí­ndrome conocido como "tristeza", II) enanismo, acanaladuras en la madera y fruta de pequeño calibre (stem pitting, SP), y III) enanismo y amarillamiento de plantas de semilla de limonero, pomelo y NA (seedling yellows, SY). La gama de huéspedes de CTV es muy restringida y hasta hace poco no se conocí­a ningún huésped herbáceo experimental. Actualmente, se sabe que la agroinfiltración de Nicotiana benthamiana, con clones de DNA complemantario (cDNA) del aislado T36 de CTV produce la infección sistémica de la planta, acompañada de sí­ntomas similares a los inducidos en cí­tricos, si bien la infección no queda limitada al floema. El aislado T36 induce SY y SP de lima Mejicana (LM), pero no en otros huéspedes como pomelo o ND. El estudio de los determinantes genéticos responsables de la inducción del sí­ndrome de SP requerí­a desarrollar un sistema genético basado en clones agroinfecciosos de un aislado inductor de estos sí­ntomas, como el aislado español T318A. Para ello, se partió de clones de cDNA de longitud completa de T318A previamente desarrollados en el laboratorio, capaces de replicarse en hojas de N. benthamiana pero incapaces de inducir infección sistémica y que presentaban varias mutaciones en su proteína de cápsida minoritaria p27. La corrección de dichas mutaciones y la construcción de nuevos clones de longitud completa de T318A marcados con el gen gfp, mostraron una correcta replicación en hojas agroinfiltradas de N. benthamiana, pero resultaron incapaces de inducir infección sistémica en este huésped experimental. La respuesta diferencial de N. benthamiana frente a distintas cepas de CTV permite estudiar los factores implicados en la interacción virus-huésped. Se analizó la interacción de las proteí­nas virales p20 y p25 de los aislados T36 y T318A con proteí­nas de N. benthamiana utilizando un abordaje consistente en: i) la expresión transitoria de p20/p25 marcadas con una etiqueta Strep-Tag en hojas de N. benthamiana, ii) purificación de los complejos proteí­na CTV-proteína huésped y análisis interactómico de los datos, y iii) estudio de la interacción directa entre p20/p25 y proteínas seleccionadas del huésped mediante análisis del doble hibrido en levadura y complementación bimolecular de fluorescencia (BIFC). Este abordaje proteómico mostró claras diferencias entre aislados que pueden explicar, en parte, el comportamiento diferencial de los aislados T36 y T318A en dicho huésped experimental. La inducción el síndrome de decaimiento por parte de CTV ha obligado a utilizar patrones tolerantes al decaimiento. Dichos patrones son menos adecuados. Las plantas de cí­tricos propagadas sobre NA e infectadas por CTV muestran necrosis en los tubos cribosos y disminución del floema funcional. Éstos desórdenes podrí­an ser consecuencia de la activación de los mecanismos de defensa como la reacción de hipersensibilidad desencadenada por la ruta del ácido salicí­lico o el silenciamiento génico mediado por RNA (post-transcriptional gene silencing, PTGS). Con el objetivo de avanzar en el mecanismo molecular de la resistencia del NA a la infección por CTV, se estudió el papel de diferentes genes de la planta implicados en las rutas mediante el uso de un vector viral basado en el genoma del virus del manchado foliar de los cítricos (citrus leaf blotch virus, CLBV). El silenciamiento génico de las rutas del AS o del PTGS en plantas NA y la inoculación de tres aislados de CTV patogénicamente diferentes mostró la implicación de ambas rutas en la defensa del NA frente a CTV.
Tristeza is the most important viral disease affecting citrus plants and Citrus tristeza virus (CTV) is the causal agent of this disease. CTV induces at least one of this syndromes: I) decline and death of sweet orange (SwO), grapefruits and mandarin trees grafted on sour orange (SO) rootstock, this syndromes is known as "tristeza", II) stunting, stem pitting (SP) and small fruits, and III) stunting and leaf chlorosis of lemon, grapefruit and SO seedlings (seedling yellows, SY). The host range of CTV is restricted and until recently no experimental herbaceous host was known. The agroinoculation Nicotiana benthamiana with clones of complementary DNA (cDNA) from the CTV isolate T36 cause the systemic infection of the plant and similar symptoms to those observed in citrus, although the infection is not limited to the phloem. T36 isolate induces SY and SP of Mexican lime (ML), but not in other hosts such as grapefruit and SwO. Therefore, to study the genetic determinants responsible of the SP syndrome induction was necessary to develop a genetic system based on agroinoculated clones from an isolate able to induce these symptoms, such as the Spanish isolate T318A. To do this, full length cDNA clones from T318A were obtained. They are able to replicate in N. benthamiana leaves but unable to induce systemic infection and showed several mutations in their protein of the minor coat, p27. The correction of these mutations and the construction of new clones of complete length from T318A labeled with the gfp gene, showed a proper replication in agroinoculated leaves of N. benthamiana, but they were still unable to induce systemic infection in this experimental host. The differential response of N. benthamiana to different CTV strains allows the study of the potential factors involved in the virus-host interaction. The aim of this work was study the interaction between the viral proteins p20 and p25 from the isolates T36 and T318A with N. benthamiana proteins with an analysis consisted in: I) the transitory expression of p20/p25 fused to Strep-Tag in N. benthamiana leaves, II) purification of the CTV protein-host protein complex and interatomic analysis of the data, and III) the study of the direct interaction between p20/p25 and selected plant proteins by the analysis of the double hybrid in yeast and bimolecular complementation of fluorescence (BIFC). The proteomic analysis showed strong differences between isolates that may partially explain the differential behavior of the T36 and T318A isolates in this experimental host. The induction of decline syndrome by CTV in citrus has leaded the use of tolerant rootstocks to decline. However, the use of such rootstocks is less suitable. Citrus plants propagated on SO rootstock and infected by CTV show phloem necrosis below the bud union that reduces the flow of carbohydrates to the roots. These symptoms may be a consequence of the activation of defense pathways in the plant, such as the hypersensitive reaction, hormone salicylic acid (SA) pathways or the RNA mediated post-transcriptional gene silencing (PTGS). Their relation is essential to know their implication in the decline. Therefore, the role of different genes involved in SA and PTGS has been studied by the silencing of plant genes using a viral vector (VIGS) based in the genome of the citrus leaf blotch virus (CLBV). The gene silencing of the SA and PTGS in SO and the inoculation of three different pathogenicity CTV isolates showed that both pathways are involved in the SO defense against CTV. The analysis of the proteins p20, p23 and p25 as possible suppressors of the AS indicating that the more virulent CTV isolates possess the more powerful suppressors.
La Tristesa és la malaltia viral més greu del cultiu dels cítrics. CTV induïx un o més de les sí­ndromes següents: I) decaïment i mort de taronger dolç§ (ND), pomelo i mandariner empeltats sobre el patró taronger amarg (NA), sí­ndrome conegut com "Tristesa", II) nanisme, estries en la fusta i fruita de xicotet calibre (SP) i III) nanisme i tonalitat groguenta de plantes de llavor de llimera, pomelo i taronger amarg (SY). El rang d'hostes de CTV és molt restringit i fins fa poc no es coneixia cap hoste herbaci experimental. Actualment es sap que la infecciò sistèmica en Nicotiana benthamiana amb clons de DNA complementari (cDNA) de l`aïllat de T36 provoca la infecció sistemàtica de la planta, acompanyada de síntomes similars als induïts en cí­trics, si be la infecció no queda llimitada al floema. L' aïllat T36 induïx SY i estries en la fusta de Llima Mexicana (LM), però no en altres hostes com a pomelo, ND o NA, l'estudi dels determinants genètics responsables de la inducció de la síndrome de SP requeria desenvolupar un sistema genètic basat en clons agroinfecciosos d'un aïllat inductor d'estos sí­mptomes, com l'aïllat espanyol T318A. Per a això, es va partir de clons de cDNA longitud completa de T318A prèviament desenvolupats al laboratori, capaços de replicar-se en fulls de N. benthamiana però incapaços d'induir infecció sistèmica i que presentaven varies mutacions en la seua proteïna de càpsida minoritatia p27. La correcció d`aquestes mutacions i la construcció de nous clons T318A de longitud completa marcats amb el gen gfp, van mostrar una correcta replicació en fulls agroinfiltradas de N. benthamiana però van resultar incapaços d'induir infecció sistèmica en aquest hoste experimental. La resposta diferencial dependent d'aïllat en N. benthamiana front CTV permet estudiar els possibles factors de la interacció virus- hoste. Es va dur a terme l'estudi de la funció de les proteínes virals p20 i p25 dels aïllats T36 i T318A amb proteïnes de N. benthamiana utilitzant un abordatge consistent en: i) l' expressió transitòria de les dues proteïnes p20/p25 marcades amb una etiqueta Strep-Tag en fulls de N. benthamiana, ii) purificació dels complexos proteïna CTV-proteïna hoste i anàlisi interactómic de les dades, i iii) estudi de la interacció directa per mitjà  de doble híbrid en llevat i complementació bimolecular de fluorescència (BIFC) de les proteïnes virals i determinades proteïnes de N. benthamiana. Aquest abordatge proteòmic va mostrar clares diferències entre aïllats que poden explicar el comportament diferencial dels aïllats T36 i T318A en aquest hoste experimental. La inducció de la sí­ndrome de decaïment per part de CTV en cí­trics ha obligat la utilització de patrons tolerants al decaïment. No obstant, aquestos patrons són agronòmicament menys adequats. Les plantes de cítrics propagades sobre NA i infectades por CTV mostren necrosi als tubs cribosos i disminució del floema funcional. Aquestos sí­mptomes poden ser conseqüència de l'activació de les rutes de defensa de la planta com la reacció d'hipersensibilitat, desencadenada per la ruta de l'àcid salicí­lic o el silenciamient gènic mediat per RNA (PTGS). Amb l'objectiu d'analitzar la implicació d¿aquestes rutes en la defensa, es va estudiar el paper de diferents gens implicats en la ruta de l'AS i del PTGS per mitjà  del silenciamient gènic induït per virus basat en el genoma del tacat foliar dels cítrics (CLBV). El silenciamient gènic de les rutes AS o PTGS en plantes NA i la inoculació de tres aïllats de CTV patogènicament diferents va mostrar la implicació de les dues rutes en la defensa del NA front CTV. L'analisis de les proteïnes p20, p23 i p25 com a possibles supressors de la ruta de l'AS va indicar que els aïllats més virulents de CTV posseïxen supressors més potents.
Gómez Muñoz, N. (2017). ESTUDIO DE LA INTERACCIÓN DIFERENCIAL ENTRE AISLADOS DEL VIRUS DE LA TRISTEZA DE LOS CÍTRICOS (CTV) Y SUS HUÉSPEDES [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/94624
TESIS

El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para intentar silenciar su expresión en células infectadas. Se ha observado resistencia completa a la infección viral en tres líneas transgénicas, manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV. Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados, lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en lima Mexicana.
Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime.
Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631
TESIS

Aunque en España los aislados más comunes de CTV son poco agresivos, en otros países suelen predominar aislados más virulentos que producen los síndromes de amarilleo (seedling yellows, SY) y acanaladuras en la madera (stem pitting, SP) en pomelo o naranjo dulce que conllevan a una disminución en la producción y calidad de los frutos. Por ello, el objetivo general de esta tesis ha sido el desarrollo de herramientas para controlar los aislados más virulentos de CTV. Para poner a punto un sistema genético eficaz que permitiera identificar los determinantes de patogenicidad de los síntomas de SY y SP se utilizó como base el aislado agresivo de origen español T318A (inductor de SY y SP), que ha sido caracterizado biológica y molecularmente. Una peculiaridad de este aislado es la presencia en su población de RNAs defectivos de gran tamaño que poseen capacidad de replicación autónoma que podrían servir como minireplicones naturales para el desarrollo de un sistema genético. Para poder evaluar la eficacia replicativa de los futuros clones de cDNA se desarrolló un protocolo de RT-PCR cuantitativa a tiempo real con SYBR Green que permite la detección y cuantificación de forma fiable de CTV en distintos tejidos y especies huésped y ha permitido identificar correctamente el 91% de los aislados como virulentos (inductores de SY, SP o ambos) o no virulentos por el valor de la temperatura de fusión (Tm) de sus productos de amplificación. A su vez, para identificar aislados potencialmente peligrosos en campo (inductores de SP en pomelo o naranjo dulce) se desarrolló un nuevo protocolo de RT-PCR a tiempo real con tres sondas Taqman tipo LNA, que permiten la detección y cuantificación de variantes de secuencia características de aislados no virulentos, virulentos inductores de SP y virulentos tipo T36 (inducen SY pero no SP). Este nuevo protocolo se aplicó al análisis de la población viral de plantas co-inoculadas con un aislado no virulento y otro inductor de SP para estudiar la capacidad invasiva y de acumulación de cada aislado. Por último, en el desarrollo de un sistema genético basado en T318A se ensayó en primer lugar su capacidad para replicarse en protoplastos de Nicotiana benthamiana. La construcción de un cDNA infeccioso del genoma completo de T318A se inició construyendo minireplicones basados en algunos de sus RNAs defectivos de gran tamaño, colocados bajo la acción del promotor 35S en un vector binario y con la pauta de la ORF 1a interrumpida por un intrón para reducir su toxicidad en bacterias. La agroinfiltración de estos clones dio lugar a una replicación eficiente en hojas de N. benthamiana y a partir de los minireplicones que más se acumulaban se obtuvieron clones de longitud completa insertándoles la región central del genoma que les faltaba. Aunque todos los clones de longitud completa se replicaron en hojas agroinfiltradas de N. benthamiana la pauta de acumulación del RNA genómico de T318A sugiere que este aislado no se mueve a células vecinas ni produce una infección sistémica.
The common CTV isolates in Spain are not very aggressive and they only produce decline of varieties grafted on sour orange, while in other countries more virulent isolates that produce the seedling yellows (SY) and stem pitting syndromes (SP) in grapefruit or sweet orange prevail with independence of the rootstock used. The general objective of this thesis was the development of tools to control the most virulent isolates of CTV. To accomplish this, the development of an effective genetic system that allow the characterization of the genetic determinants of the symptoms of SY and SP has begun. To set up an effective genetic system that permit the identification of the pathogenicity determinants of the symptoms of SY and SP the aggressive Spanish isolate T318A (inductor of SY and SP) was used, that was characterized biological and molecularly. A peculiarity of this isolate is the presence in its population of defective RNAs of high size that possess capacity of autonomous replication. To evaluate the replicative ability of the cDNA clones it was developed a real-time RT-PCR assay using SYBR Green for specific and reliable quantitative detection of CTV in different citrus species and tissues infected with pathogenically distinct CTV isolates sampled from plants growing in the greenhouse or in the field. Also, to identify isolates potentially dangerous in the field (able to induce SP in grapefruit or sweet orange trees) a new protocol of real time RT-PCR was developed with three Taqman LNA probes that allow the detection and quantification of characteristic sequence variants of each of the three main CTV groups observed, that include mild, severe SP, and T36-like isolates, respectively (SY but non SP). Finally, in the development of a genetic system based on T318A, the agroinfiltration of these clones provided an efficient replication in leaves of Nicotiana benthamiana although the accumulation of genomic RNA of T318A suggests that this isolate does not move to neighbouring cells neither it produces a systemic infection.

No Brasil a citricultura está entre as culturas de maior importância. A produtividade dessa cultura no país ainda é considerada baixa e esse fato se deve, em parte, a diversas pragas e doenças. Dentre as doenças, tem-se a tristeza, causada pelo Citrus tristeza virus (CTV). Esse patógeno também pode estar relacionado com outra importante doença da cultura, a morte súbita dos citros (MSC). Com isso, o CTV ganhou ainda maior expressão. Uma alternativa para controlar viroses de plantas é a obtenção de plantas transgênicas resistentes a esses patógenos. Este trabalho objetivou caracterizar com análise molecular e avaliar a resistência ao CTV de plantas transgênicas de laranja Valência (Citrus sinensis L. Osbeck), contendo fragmentos do genoma do CTV, em três construções gênicas diferentes. A transgenia das plantas foi confirmada por análises de Southern blot. A transcrição do transgene foi avaliada por RT-PCR. O material foi inoculado com duas borbulhas infectadas pelo isolado Pêra- IAC, enxertadas no porta-enxerto abaixo do ponto de enxertia da copa transgênica, e pelo vetor Toxoptera citricida infectado. Após quatro semanas da inoculação, para avaliar a resistência ao vírus, brotações da copa transgênica foram submetidas ao teste de ELISA sanduíche indireto com anticorpo monoclonal contra a proteína da capa protéica do CTV. Os resultados indicaram variação na resistência à translocação do vírus nas diferentes construções transgênicas utilizadas e entre clones de uma mesma planta. Todas as linhagens transgênicas inoculadas indicaram a presença do vírus em pelo menos uma das três repetições avaliadas, quando inoculadas por enxertia. Quando inoculadas pelo vetor algumas plantas apresentaram todos os seus clones com baixos valores de absorbância, indicando uma possível resistência ao patógeno.
In Brazil, citrus is one of the most important cultures. The productivity of this culture in the country is still considered low and this fact is due to several pests and diseases that affect the crop. Among the diseases there is the tristeza, caused by Citrus tristeza virus (CTV). This pathogen can also be related with another important disease, the citrus sudden death. Therefore, CTV acquired much more significance. This work aimed to characterize with molecular analysis and to evaluate the resistance to CTV of transgenic Valência plants (Citrus sinensis L. Osbeck), containing genomic fragments of CTV, in three different transgenic constructs. The plants were confirmed as transgenic by Southern blot. The transcription of the transgene was evaluated by RT-PCR. The transgenic plants were challenged with a weak strain of CTV, CTV-IAC, by bud inoculation with two infected bubbles, and by the infected vector Toxoptera citricida. After four weeks of inoculation, the evaluation of viral replication in the transgenic seious was done by ELISA indirect sandwich with monoclonal antibody against the CTV coat protein. The results indicated variation of the resistance to the translocation of the virus between the different transgenic constructs used and between clones of the same plant. All the inoculated plants indicated the presence of the virus in, at least, one of the three evaluated clones, when inoculated by grafting. When inoculated by the vector some plants had all their clones with low values of virus, indicating a possible resistance to the pathogen.

This dissertation profiles the children's television industry in Australia; examines the relationship between government cultural policy objectives and television industry production practices; and explores the complexities of regulating and producing cultural content for child audiences. The research conducted between 1997 and 2002 confirms that children's television is a highly competitive business dependent on government regulatory mechanisms and support for its existence. For example, the Australian Broadcasting Authority's retaining of mandatory program standards for children's programs to date, is evidence of the government's continued recognition of the conflict between broadcasters' commercial imperatives and the public-interest. As a consequence, the industry is on the one hand insistent on the government continuing to play a role in ensuring and sustaining CTV - however, on the other hand, CTV producers resent the restrictions on creativity and innovation they believe result from the use of regulatory instruments such as the Children's Television Standards (CTS). In fact, as this dissertation details, the ABA's intended policy outcomes are inevitably coupled with unintended outcomes and little new or innovative policy development has occurred. The dissertation begins with an investigation into the social, cultural and ideological construction of childhood within an historical and institutional context. I do this in order to explore how children have been defined, constructed and managed as a cultural group and television audience. From this investigation, I then map the development of children television policy and provide examples of how 'the child' is a consistent and controversial site of tension within policy debate. I then introduce and analyse a selection of established, establishing and aspiring CTV production companies and producers. Drawing on interviews conducted, production companies profiled and policy documents analysed, I conclude by identit~'ing ten key issues that have impacted, and continue to impact, on the production of children's television programming in Australia. In addressing issues of industry development, the question this dissertation confronts is not whether to continue to regulate or not, but rather, how best to regulate. That is, it explores the complexities of supporting, sustaining and developing the CTV industry in ways which also allows innovative and creative programming. This exploration is done within the context of a broadcasting industry currently in transition from analogue to digital. As communications and broadcasting technologies converge, instruments of regulation - such as quotas designed around the characteristics of analogue systems of broadcasting - are being compromised. The ways in which children use television, and the ways in which the CTV producers create content, are being transformed. The ten key issues identified in this dissertation, I propose, are crucial to industry development and policy debate about the future of children's television in Australia. In integrating the study of policy with the study of production, I have given prominence to the opinions and experiences of those working in the industry. In doing so, this dissertation contributes to the growing body of work in Australia which incorporates industry with cultural analysis, and which includes the voices of the content providers.

This dissertation profiles the children's television industry in Australia; examines the relationship between government cultural policy objectives and television industry production practices; and explores the complexities of regulating and producing cultural content for child audiences. The research conducted between 1997 and 2002 confirms that children's television is a highly competitive business dependent on government regulatory mechanisms and support for its existence. For example, the Australian Broadcasting Authority's retaining of mandatory program standards for children's programs to date, is evidence of the government's continued recognition of the conflict between broadcasters' commercial imperatives and the public-interest. As a consequence, the industry is on the one hand insistent on the government continuing to play a role in ensuring and sustaining CTV - however, on the other hand, CTV producers resent the restrictions on creativity and innovation they believe result from the use of regulatory instruments such as the Children's Television Standards (CTS). In fact, as this dissertation details, the ABA's intended policy outcomes are inevitably coupled with unintended outcomes and little new or innovative policy development has occurred. The dissertation begins with an investigation into the social, cultural and ideological construction of childhood within an historical and institutional context. I do this in order to explore how children have been defined, constructed and managed as a cultural group and television audience. From this investigation, I then map the development of children television policy and provide examples of how 'the child' is a consistent and controversial site of tension within policy debate. I then introduce and analyse a selection of established, establishing and aspiring CTV production companies and producers. Drawing on interviews conducted, production companies profiled and policy documents analysed, I conclude by identit~'ing ten key issues that have impacted, and continue to impact, on the production of children's television programming in Australia. In addressing issues of industry development, the question this dissertation confronts is not whether to continue to regulate or not, but rather, how best to regulate. That is, it explores the complexities of supporting, sustaining and developing the CTV industry in ways which also allows innovative and creative programming. This exploration is done within the context of a broadcasting industry currently in transition from analogue to digital. As communications and broadcasting technologies converge, instruments of regulation - such as quotas designed around the characteristics of analogue systems of broadcasting - are being compromised. The ways in which children use television, and the ways in which the CTV producers create content, are being transformed. The ten key issues identified in this dissertation, I propose, are crucial to industry development and policy debate about the future of children's television in Australia. In integrating the study of policy with the study of production, I have given prominence to the opinions and experiences of those working in the industry. In doing so, this dissertation contributes to the growing body of work in Australia which incorporates industry with cultural analysis, and which includes the voices of the content providers.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Arts, Media and Culture
Full Text

En la presente tesis se ha pretendido avanzar en el conocimiento de la resistencia al virus de la tristeza de los cítricos (CTV) con el propósito de diseñar estrategias de mejora más eficientes.En este trabajo se ha obtenido un nuevo grupo de marcadores moleculares derivados de retrotransposones del tipo gypsy y se ha analizado la presencia, heterogeneidad y distribución de dichos elementos en especies de los géneros Citrus y Poncirus. A partir de una variedad de mandarino clementino se han aislado ocho clones que contienen una parte de la región codificante POL de los retrotransposones tipo gypsy. El análisis de sus secuencias parece indicar que cuatro de estos clones corresponden a elementos potencialmente activos, puesto que presentan intactos, sin codones de paro ni desplazamiento de la pauta de lectura correcta, todos los motivos conservados indispensables para la retrotransposición. En la mejora genética de cítricos uno de los principales objetivos es la resistencia a CTV. La tristeza de los cítricos es la principal virosis que afecta al cultivo de los cítricos en el mundo. La obtención de plantas resistentes a su agente causal (CTV) sería la forma más adecuada de combatir la enfermedad. P. trifoliata es una especie resistente al virus y sexualmente compatible con las especies del género Citrus, por lo que tradicionalmente en los programas de mejora de cítricos se le ha considerado como la mejor candidata para transferir su resistencia a los cítricos cultivados. Varios estudios anteriores han definido la resistencia de P. trifoliata a CTV como un carácter controlado por un único gen (Ctv-R) con el objetivo de obtener cítricos comerciales resistentes al virus a partir de la transformación genética con el gen de resistencia a CTV. En la presente tesis se ha saturado con distintos tipos de marcadores moleculares la región donde previamente se había situado Ctv-R. Para ello se han utilizado dos familias segregantes derivadas de P. trifoliata, una por autopolinización y la otra por cruce con C. aurantium (naranjo amargo), un patrón bien adaptado a las condiciones semi-áridas típicas de la cuenca mediterránea. El seguimiento de la infección con CTV en la familia AxPa ha proporcionado resultados sorprendentes. Amargo resulta tan resistente al aislado del virus ensayado (T-346) como P. trifoliata. Los mapas saturados resultantes de este estudio se han utilizado para localizar la resistencia al virus bajo la hipótesis de transmisión monogénica. El análisis genético de la interacción virus-planta en la familia AxPa tras la infección crónica con CTV ha revelado cinco tipos distintos de interacción lo cual es incompatible con la hipótesis de un sólo gen de resistencia. Los resultados obtenidos indican que la transferencia del gen Ctv-R de P. trifoliata al naranjo amargo podría no evitar los síntomas típicos de decaimiento ("decline") en naranjo dulce injertado sobre este nuevo patrón.Los mapas saturados obtenidos con anterioridad en este trabajo se han utilizado para localizar la resistencia a CTV considerándola como carácter cuantitativo (acumulación de virus) mediante análisis de QTLs ("quantitative trait loci"), evitando así la asunción de control monogénico de la resistencia. Los resultados revelan la presencia de tres QTLs de resistencia principales, dos en P. trifoliata y uno en naranjo amargo, en la región donde se había localizado Ctv-R en otras progenies. Además, se han detectado otros QTLs de acumulación donde se localizan genes de menor efecto. También se han detectado interacciones epistáticas implicadas en el control genético del carácter.
Citrus is an extensively apomictic genus and transposable elements might be importantly involved in its genetic instability and genome evolution. The presence of gypsy-like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus have been investigated. Eight clones containing part of the POL coding region of gypsy- like retrotransposons have been isolated from a commercial variety of C. clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. Nested copies of gypsy-like elements are scattered along the Citrus and Poncirus genomes. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently providing a new, complementary set of molecular markers now available in Citrus to study and follow genetic variability, specially for disease resistance. Several studies have reported markers linked to a putative resistance gene from Poncirus trifoliata (Ctv-R) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (CTV). Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange, the well-adapted citrus rootstock to arid and semi-arid areas, were used for linkage group 4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. Surprisingly, sour orange resulted as resistant to the CTV isolate tested as P. trifoliata was. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction which is not compatible with the hypothesis of a single gene controlling resistance. Transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees. Resistance against CTV was analysed as a quantitative trait (CTV accumulation) by QTL analysis to avoid the assumption of monogenic control. Three major resistance QTLs were detected. Up to 5 minor QTLs were also detected.

Citrus tristeza virus (CTV) ocorre em quase todas as áreas produtoras de citros do mundo. O controle da doença se baseia, principalmente, no uso de porta-enxertos tolerantes e na premunização das copas. A obtenção de copas de laranjas doces ou de porta-enxerto de laranja azeda transgênicos resistentes ao CTV permitiria retornar a um uso mais intensivo deste excelente porta-enxerto. Com isso, esse trabalho buscou avaliar linhagens transgênicas de laranja doce (Citrus sinensis) e obter plantas transgênicas de laranja azeda (Citrus aurantium) para a resistência ao CTV, a fim de oferecer uma outra alternativa para o controle desta doença em citros. Foram avaliadas plantas transgênicas de laranja doce cv. Valência e cv. Hamlin contendo três diferentes construções gênicas. Uma contendo uma sequência sense (684 pb) do gene da capa protéica do CTV (pCTV-CP), outra contendo uma sequência conservada (559 pb) do CTV (pCTV-SC) e uma do tipo hairpin, contendo sequências sense e antisense do gene da capa protéica separadas por um íntron (pCTV-dsCP). Dez linhagens transgênicas de cada construção gênica e de cada cultivar foram previamente confirmadas por análises de Southern blot e RT-PCR, totalizando 60 linhagens transgênicas. Tais linhagens foram clonadas e enxertadas sobre limão Cravo (C. limonia) e laranja azeda (C. aurantium), totalizando 360 plantas. Essas plantas, juntamente com plantas não transgênicas utilizadas como controle, foram inoculadas com o CTV por meio de Toxoptera citricida virulífero. As técnicas de ELISA indireto utilizando anticorpo monoclonal contra a capa protéica do CTV ou de Real-time PCR utilizando primers amplificadores dos genes p20 e p23 do genoma do CTV foram utilizadas para detectar o vírus nas plantas avaliadas, 4 semanas após as inoculações. Ocorreu variação na resistência ao vírus nas diferentes construções transgênicas utilizadas e entre clones de uma mesma planta. Alguns clones não foram infectados com o vírus mesmo após a quarta inoculação, indicando uma possível resistência ao patógeno. Um total de 30 experimentos de transformação genética de laranja azeda foram realizados, utilizando como explantes segmentos internodal, de epicótilo e de cotilédone associado ao hipocótilo. O teste de GUS permitiu a identificação de duas gemas com reação positiva (0,13% de eficiência de transformação). Tais gemas foram enxertadas in vitro sobre citrange Carrizo, mas apenas uma gema se desenvolveu. A planta obtida foi aclimatizada em casa-de-vegetação.
Citrus tristeza virus (CTV) occurs in almost all citrus-growing areas of the world. Control of citrus tristeza relies mainly on the use of tolerant rootstocks and scion cross protection. Obtaining transgenic sweet oranges cultivars or sour orange resistant to CTV would allow a better use of this excellent rootstock. This way, the aim of this work was to evaluate transgenic sweet orange (Citrus sinensis) lines and to obtain transgenic sour orange (Citrus aurantium) for the resistance to CTV, in order to offer another alternative for the control of the disease in citrus. Transgenic sweet orange cv. Valencia and cv. Hamlin containing three different genetic constructs were evaluated. One gene construct contains a sense sequence (684 pb) of the coat protein gene of CTV (pCTV-CP), another contains a conserved sequence (559 pb) of CTV (pCTV-SC), and the last one a hairpin type, containing the sense and antisense sequences of the coat protein gene separated by an intron (pCTV-dsCP). Ten transgenic lines of each gene construct and each cultivar were previously confirmed by Southern blot and RT-PCR analysis, totalizing 60 transgenic lines. These lines were cloned and grafted into C. limonia and into C. aurantium, totaling 360 plants. The plants, along with non-transgenic plants used as control, were challenged four times with the CTV by means of viruliferous Toxoptera citricida. Indirect ELISA using monoclonal antibody against the CTV coat protein or the Real-time PCR using primers to amplify the CTV genes p20 and p23 were used to detect the virus in the tested plants, 4 weeks after inoculation. Variation in the virus resistance was observed among different transgenic constructs and different clones of the same plant. Some clones were not infected with the virus even after the fourth inoculation, indicating a possible resistance to the pathogen. A total of 30 genetic transformation experiments of sour orange were performed, using as explants internodal segments, epicotyl segments and cotyledon fragment with hypocotyl attached. GUS reaction detected two shoots positive (transformation efficiency of 0,13%). These shoots were in vitro grafted in Carrizo citrange, but only one shoot developed. The plant obtained was acclimatized in greenhouse.

Introduction. Des traitements efficaces permettent la prise en charge des personnes vivant avec le VIH (PVVIH) et la prévention. Cependant, seulement 55% des PVVIH connaissent leur statut et ce taux est encore plus faible pour les pays de l’Afrique de l’Ouest et du Centre (35%). Afin d’accroître l’offre de Conseil et Test pour le VIH (CTV), l’OMS a publié en juillet 2015 un guide consolidé basé sur des études, dont certaines ont été réalisées en Afrique Subsaharienne. Et très peu d’entre elles concernent les pays à faible prévalence du VIH, notamment l’Afrique de l’Ouest francophone. Cette thèse a pour objectif général d’analyser les enjeux et les limites des politiques et programmes de CTV dans les pays africains de basse prévalence VIH, à partir de la situation du Burkina Faso, et de proposer de nouvelles mesures pour développer l’offre de CTV.Méthodes. Deux études ont été conduites. La première portait sur les motivations et les obstacles à la pratique du test VIH. Elle a été menée dans le cadre du projet « Multi-country African Testing and Counselling for HIV » (MATCH) portant sur quatre pays (Burkina Faso, Kenya, Malawi, Ouganda). Au Burkina Faso, l’étude a été conduite en 2008-2009 en milieu urbain (Ouagadougou) et rural (Dédougou) dans des sites sélectionnés selon leur niveau de fréquentation. Des approches quantitatives et qualitatives ont été utilisées.La deuxième étude a été conduite en 2015 auprès des acteurs et des décideurs du CTV au niveau national, et visait à analyser leurs perceptions des directives publiées par l’OMS en 2015. Un outil de collecte des données expliquant les changements a diffusé par voie électronique. Les réponses ont été analysées de manière quantitative et qualitative.Résultats. L’offre du CTV est basée au Burkina Faso sur des documents validés en 2008.L’analyse de l’utilisation du test à l’initiative du client a montré que les femmes étaient les plus nombreuses (58,5%). Cependant les hommes (p=0,02), les 18-34 ans (p=0,01), et les plus scolarisés (p=0,001) semblaient utiliser plus précocement les services.. En analyse multivariée, ces catégories utilisaient plus les campagnes. Les signes ou symptômes liés au VIH motivaient le test chez les femmes (p=0,008), les 35 ans et plus (p<0,001) et les non scolarisés (p<0,001) qui sollicitaient plus le test en sites fixes. L’utilisation du CTV pendant la campagne était associée au désir de connaître le statut (p<0,001), tandis qu’en dehors des campagnes, l'état de santé de l’utilisateur, la maladie ou le décès du partenaire était le principal motif (p=0,001). Aussi 61% des utilisateurs avaient réalisé 2 tests et plus. Dans une analyse multivariée, l’utilisation répétée du CTV par les personnes séronégatives était associée à la scolarisation (au moins le secondaire), au jeune âge et pour les PVVIH à la résidence en milieu urbain.Les prestataires déclaraient être confrontés à des difficultés logistiques et matérielles pour offrir adéquatement le CTV. Il en résultait une faible qualité des services, notamment un conseil post test dispensé partiellement, et une faible référence des PVVIH. Celles-ci avaient un vécu de stigmatisation élevé, soit 46% de stigmatisation interne, 40% dans les relations interpersonnelles et 11% dans les services de santé. Les décideurs et acteurs ont trouvé la plupart des directives de 2015 pertinentes, mais sont pessimistes sur leur faisabilité.Conclusion. Ce travail a identifié les limites du CTV au Burkina Faso et donne des éléments significatifs pour les pays africains de basse prévalence. Dans un contexte de raréfaction des ressources, l’accès équitable au CTV nécessite l’identification de stratégies innovantes. Le renforcement des capacités des prestataires pour une offre globale de services de qualité est nécessaire. La lutte contre la stigmatisation devrait être intensifiée. La prise en compte des avis des experts permettra la révision des documents nationaux et leur adaptation selon les directives OMS
Background. Treatments are effective for people living with HIVAIDS (PLWHA) care and prevention. However, only 55% of PLWHA are aware of their status. This rate is lower in Central and West Africa (35%). In July 2015, WHO published new guidelines on HIV testing. In Sub-Saharan Africa, there are many studies which results are used to define policies and guidelines on HIV testing at the international level. Few of them are implemented in low HIV prevalence countries, notably French-Speaking West Africa. The overall objective of this thesis is to analyze the challenges and limitations of HIV testing and counselling (HTC) policies and programs in low prevalence countries, over the situation in Burkina Faso and propose new measures to increase the access to HTC services.Methods. Two studies have been conducted. The first one was carried out in the « Multi-country African Testing and Counselling for HIV » (MATCH) project which was implemented in four countries (Burkina Faso, Kenya, Malawi, Uganda). It aimed to analyze the motivations and barriers to HTC services practices by users, non-users and providers. In Burkina Faso, the study was carried out in Urban (Ouagadougou) and rural (Dédougou) areas in 2008-2009. In each locality, study sites (client initiated testing and provider initiated testing sites) were chosen, given the level of utilization. Quantitative and qualitative methods were used. The second study was conducted in 2015, with HTC providers and decision makers at national level. The objective was to analyze their perceptions on WHO 2015 guidelines. A data collection tool explaining the changes introduced in the guidelines has been designed and transmitted via electronic means. Their opinions were analyzed.Results. Guidelines for HTC in Burkina Faso were from 2008. In client initiated testing sites, there were more women (58.5%). However, men (p=0.02), 18-34 years old (p=0.01), and the more educated ones (p=0.001) appeared to have used early services. In multivariate analysis, those categories used often campaigns. Women (p=0.008), 35 years of age and over (p<0.001) and less educated people (p<0.001) sought more often the test in fixed sites. The use of HTC services during campaigns is associated with the desire to know one's HIV status (p<0.01), while outside of campaigns, the health status of the user, the illness or the partner's death was the main concern (p=0.001). Campaigns are associated with the hope of knowing one’s HIV status (p<0.001). There were 61% of users who were repeat testers (2 or more tests). In a multivariate analysis, repeat testing for HIV negative people was associated with higher education, young age and for PLWHA living in urban areas. HTC Providers declared that they faced logistic and material challenges. It resulted in a low quality of services, in particular post-test counselling sessions that were partially done and a low effective reference of PLWHA towards care services. HIV stigma was found to be very high (46% of PLWHA faced internal stigma, 40% of interpersonal stigma, and 11% in health services). Decision-makers and providers have found most of the 2015 guidelines relevant, but were pessimist about their feasibility.Conclusion. This work has identified limitations of HTC at the individual, community, health services and institutional levels in Burkina Faso, and provides significant elements for African low prevalence countries. Given the scarcity of resources, there is a need for innovative strategies for equitable access to HTC, in order to attract more men, and test earlier women, less educated and 34 years or older. Strengthening the capacity of service providers to include a comprehensive range of quality services is necessary. All the aforementioned should be supplemented by the fight against stigma. Paying attention to national HTC experts’ opinion will help for national guidelines review and adapt them to WHO guidelines

Le virus de la tristeza (ctv) provoque une des maladies les plus destructrices des agrumes. Sur l'ile de la reunion, la presence de souches virales severes limite le developpement des especes directement sensibles au virus. L'indexage biologique d'une vingtaine d'isolats locaux du virus sur cinq plantes hotes a mis en evidence des differences qualitatives et quantitatives de pouvoir pathogene entre isolats, allant de modere a severe. Cependant tous les isolats etudies apparaissent homogenes quant a leur reaction avec dix anticorps monoclonaux. Une faible variabilite du gene de capside a ete observee par restriction avec les enzymes eco ri, hae iii et rsa i. Les mesures de vigueur ont montree l'action premunisante de plusieurs isolats locaux sur combava (citrus hystrix). La transmission du ctv sur le mode semi-persistant par toxoptera citricidus a ete confirmee. Parmi les especes potentiellement hotes du virus (rutacees et passiflores) les citrus representent le reservoir principal de l'agent pathogene. Un delai minimum de deux a quatre mois est necessaire a la detection du ctv par le test elisa dans l'ensemble de la frondaison de jeunes plants greffes. L'analyse spatio-temporelle, a l'aide d'anticorps monoclonaux, des cinetiques de contamination de neuf vergers plantes indemnes a montre l'activite vectrice elevee des pucerons sur l'ile. En effet, malgre les variations enregistrees entre vergers d'especes ou d'implantations differentes, toutes les parcelles ont ete entierement contaminees avant leur entree en production (3eme annee). Neanmoins, des perspectives interessantes de diminution des contaminations par vecteur grace a l'emploi du paillage plastique noir ont ete mises en evidence. Parmi les principaux vecteurs connus du ctv presents a la reunion, t. Citricidus est l'espece qui assure l'essentiel des contaminations en raison de l'abondance des populations infestant les agrumes, de sa meilleure efficacite intrinseque de transmission et de sa gamme de plantes hotes restreinte aux rutacees

Thesis (Ph. D.)--Ohio State University, 2004.
Document formatted into pages; contains xix, 219 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Oct. 29.

El virus de la tristeza de los cítricos (CTV) ha causado millonarias pérdidas a la citricultura a nivel mundial. En Chile, la situación real era desconocida aunque se había reportado su presencia en limoneros Meyer, los que fueron erradicados. El objetivo de esta tesis fue analizar la situación actual de CTV en Chile, realizar una amplia prospección y estimar su incidencia, estudiar su epidemiología y realizar una caracterización biológica, serológica y molecular de 100 aislados chilenos de CTV representativos. Se realizó un estudio epidemiológico en seis parcelas experimentales y dos ensayos de trasnsmisión con 10 aislados representativos. CTV fue detectado en casi todas las zonas citrícolas de Chile, con una incidencia media del 0,38%. En el oasis de Pica (I Región), se observaron acanaladuras en la madera en árboles de pomelo y lima mejicana. Se detectan todas las especies vectoras de CTV, a excepción de Toxoptera citricida. En la zona central de Chile se determina la presencia mayoritaria de aislados atenuados (MCA13 negativos). En la I Región (oasis de Pica y Malilla) la presencia de aislados agresivos, siendo todos MCA13 positivos y características moleculares asociadas al tipo VT. Se demostró un aumento en la incidencia de CTV en las parcelas experimentales, salvo en un huerto de limonero. En ensayos de transmisión sólo un aislado de CTV fue transmitido con una eficiencia baja de transmisión. En base a los resultados obtenidos se dan recomencaciones para la citricultura chilena.
Besoain Canales, JA. (2008). Incidencia, caracterización y epidemiología del virus de la tristeza de los cítricos en Chile [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/2009
Palancia

Introduction: Breast cancer is the most frequently diagnosed cancer found in women across the world, with an estimated 2.3 million new cases occurring in 2020. Additionally, over 684,000 deaths annually are attributed to breast cancer across the globe, making it the most common cause of cancer-related death in women. Further, treatment of breast cancer relies heavily on whether or not the cancer cells express estrogen, progesterone, and HER 2 receptors and this expression profile is often related to how quickly the cells grow and spread. In the United States, breast cancer cells that are hormone receptor positive and HER-2 negative make up about 73% of breast cancer cases, and cells that do not express any receptor and are known as triple negative, make up around 12% of cases (American Cancer Society, 2019). With that being said, CT1 and CT3 are novel compounds that have a cytotoxic effect on cell lines representing up to 85% of all breast cancer subtypes in the United States. Methods: The leaves of Chromolaena tacotana that contains the flavonoids CT1 and CT3 were dried and placed in a soxhlet extractor using dichloromethane (CH2Cl2) to extract the chlorophyll. The flavonoids were extracted using a column chromatography eluted with trichloromethane (CHCl3), a 1:1 dilution of CHCl3:methanol and methanol, followed by isolation and purification of the compounds. Human breast cancer cell lines MCF7, MDA-MB-231, and SKBr3 were treated with CT1 and CT3 at concentrations of 5, 10, 20, 40 and 80 µM, followed by incubation for 24 hours. To assess cell viability an MTT assay was conducted by adding a 5-diphenyl-tetrazolium bromide reagent. The purple-colored formazan crystals were solubilized with acidified isopropanol, then analyzed by spectrophotometry. Results: CT1 appeared to have the most cytotoxic effects compared to CT3 on MCF7. The opposite effect was observed for SKBr3 with CT3 showing the most effects as compared to CT1. No differential effect was observed on MDA-MB-231 since both CT1 and CT3 showed similar inhibition of cell viability. Conclusions: The results from the different breast cancer cell lines SKBr3, MCF7, and MDA-MB-231 vary based on how they responded to CT1 and CT3. CT3 was more effective on SKBr3 than CT1. CT1 was more effective on MCF7 than CT3. For MDA-MB-231, both CT1 and CT3 showed similar significant cytotoxic effects. The antiproliferative effects of CT1 and CT3 appear to be concentration dependent on all cells studied. In view of the results from MDA-MB-231 triple negative breast cancer cell line, the cytotoxic effect of the flavonoids is not dependent on the presence of estrogen, progesterone, or HER2 receptors on breast cancer cells. Further studies on the mechanism of action are necessary to elucidate the molecular targets of CT1 and CT3.

A methodology for the simulation of CPV systems is presented in four distinct sections: input, optics, uncertainty and electrical output. In the input section, existing methods of describing the solar irradiation that is incident at the primary optical element of a CPV system are discussed, the inadequacies of the existing methods are explored and conditions of validity for their use drawn. An improved and spectrally extended model for a variable, spatially resolved solar image is arrived at. The model is used to analyse losses at the primary concentration device stage under varying solar profiles and air masses. A contextual analysis of an example Seattle based CPV system operating with constant solar tracking errors of 0.3-0.4° show a corresponding loss in isolation available to the optical system of 5-20%, respectively. In the optics section, an optical ray trace model is developed specifically for this work. The optical ray trace model is capable of the spectrally resolved ray tracing of all insolation input models discussed above. Plano-convex and Fresnel lenses are designed, investigated and compared using each of the insolation models described in the input section. Common CPV component material samples for the plano-convex and Fresnel lenses are analysed for their spectrally resolved optical properties. The computational expense of high resolution spatial and spectral modelling is addressed by means of a spectrally weighted banding method. The optical properties parameter spectral weighting method can be applied to any arbitrary spectral band. The bands used herein correspond to the active ranges of a typical triple-junction solar cell. Each band shows a different spectral dependency. Banded beam irradiation proportions are shown to change by as much as 10% in absolute terms within the air mass range of 1 to 3. Significant variations in spectrally banded illumination profiles are found with the extended light source insolation model. These banded variations are mostly unaccounted for with the use of approximated insolation models, further compounding the argument for extended light source Sun models in CPV system simulations. In the uncertainty section, the limitations of the manufacturing process are explored. Manufacturing tolerance errors from manufacturer datasheets are presented. These production uncertainties are used in the design of an erroneous plano-convex lens which is then analysed with the optical modelled presented in the optics section and compared to the ideal design specification. A 15% variation in maximum intensity value is found alongside a linear shift in the focal crossover point of approximately 0.2mm, although the optical efficiency of the lens remains the same. Framing manufacture errors are investigated for a square Fresnel lens system resulting in a linear shift of the focal centre of approximately 0.85mm. A process for the calculation of wind loading force on a CPV array is also presented. The process uses real 2 second resolution wind data and highlights the chaotic nature of loading force. A maximum force of 1.4kN was found on an example day for a 3m by 3m by 0.1m cuboid (i.e. CPV array); corresponding to a wind speed of approximately 13m/s, which is well within the typical operating range of a CPV tracking system. In the electrical output section, a spatially resolved solar cell model is identified and used for the investigation of solar cell performance under the inhomogeneous cell illumination profiles produced in the uncertainty section. Significant differences in the maximum power point of the cell IVs are found for the ideal and erroneous system illumination profiles. Approximately, a 15% variation is found in the plano-convex lens example, with a relative difference of 4% attributable to illumination profile distortion, and a 6% variation in the module framing component example. These results further highlight the need for the consideration of production uncertainties in CPV system simulation.

A carbon dioxide emission calculator for buildings created by the U.S.-based company CTG Energetics, Inc. and based on a Excel file has been converted to a ASP.NET / SQL Server web application. Carbon dioxide emissions are calculated using data given by the user (i.e. floor area, workdays per year) in combination with statistical data used in user-selectable presets (i.e. building type, climate zone, type of water-using fixtures). In most cases a custom value can be inserted instead of using a preset. Emissions attributable both directly and indirectly to the building such as building energy use, domestic water use, landscape/irrigation, transportation, materials used for the building/parking lot and the disposal of solid waste are calculated. The emissions can be compared with a national average and/or emissions from alternate scenarios created for the same building. The web application contains some upgrades and extra functionality that would not have been possible in Excel such as user handling.

CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO
A presente dissertação pretende descrever e analisar as potencialidades futuras oferecidas por sistemas CATV operando serviços de comunicação iterativos e de faixa larga. Neste contexto, visando determinar as configurações mais adequadas para a parte óptica da rede, são analisadas diversas alternativas de implementação envolvendo os dispositivos constituintes básicos: laser, modulador externo, EDFA, e fibra, sendo neste último caso, tanto a utilização de fibra padrão quanto a de dispersão deslocada. Dentro do espírito de serviços de faixa larga, implementa- se em laboratório um sistema WDM com duas portadoras ópticas. A primeira, em 1310 nm, é modulada em intensidade com 2.48 Gbits/s. a segunda, em 1550nm, é modulada em AM- VSB. As interferências mútuas são então medidas e confrontadas com resultados previstos teoricamente, validando este último modelo. A formulação teórica permite então prever a deterioração mutua em um WDM com duas portadoras digitais em 2.48Gbit/s. Os resultados obtidos sugerem a viabilidade deste último arranjo para portar vídeo digitalizado. Dando seqüência, são apresentadas novas soluções para compressão de vídeo, técnicas de modulação e tecnologias de transporte. Finalmente, os resultados obtidos anteriormente, tanto os práticos quanto os teóricos e os simulados, são usados para propor novas arquiteturas de redes de CATV explicitando as sugestões para o caso de redes HFC (preferencialmente destinado a operadoras de TV a cabo) e também para configurações FTTC, estas últimas, preferencialmente destinadas às operadoras telefônicas.
This work is aiming at discussing the future possibilities the CATV systems are likely to offer, concerning iterative communications and broadband services. In what concerns the system optical section, a search is carried out in order to reach suitable arrangements and configurations concerning the basic used components, namely: laser, external modulator, EDFA and the optical fiber. Both standard and dispersion shifted fiber utilization are considered. Considering broadband utilization, a practical experiment is carried out. A two carrier WDM system is tested in the laboratory. The first carrier - 1310 nm - is digitally modulated at 2.48 Gbit/s, while the second one - 1550 nm - carries an AM-VSB modulation. Crosstalk is measured and the obtained results are compared with those from a proposed theoretical model. A satisfactory agreement validates the model. Next, the same model is then used for predicting crosstalk level in another two carrier WDM, however now, with both carriers being digitally modulated at 2.48 Gbit/s. the obtained results suggest that this last arrangement is worthwhile to be used for efficiently carrying digital video. A discussion on new solutions for video compression, modulation formats and transport techniques is follwed. The work is closed with a number of new CATV network solutions being suggested. Previously obtained results and performed suggestions are used for achieving two basiv lines of applications. The first one is based on HFC arrangements, mainly conceived for cable TV operators. The second on is based on FTTH arrangements, being this last suggestion better suited for traditional telephone companies.

This thesis coinsiders the two areas of CCTV research, town centre statistical studies and public attitude surveys and how CCTV has through widespread introduction, impacted upon the concept of privacy. The concept of privacy taken by this thesis is wider that the legal definition and is most briefly but relevantly defined as the 'freedom from surveillance by closed circuit television.' Town centre studies aim to look at the effectiveness of CCTV as a method of crime control. Public attitude surveys aim to use quantitative research questionnaires to discover the opinions of CCTV users, to gauge CCTV's effect upon the fear of crime and to analyse the public's expectations and actual experiences of CCTV and to consider if the social benefit of CCTV over the threat to privacy is on balance, worth the loss in privacy. Town centre and public attitude studies are the only types of published research thus far conducted upon CCTV. Three innovative studies have attempted to disperse the view that CCTV had specific uses, benefits and limitations. These studies focused on CCTV as a benefit to retail consumerism, the attitudes of offenders and the opinions and practices of CCTV operators. This thesis considers whether the threat to privacy is greater when surveillance is operated by private security companies in quasi-public places. The thesis considers how the general public's desire to shop has increased the likelihood of their submitting to such quasi-public space surveillance in return for the benefits of consumption

Verificação de modelos é uma das mais eficientes técnicas de verificação automática de sistemas. No entanto, apesar de poder lidar com verificações complexas, as ferramentas de verificação de modelos usualmente não fornecem informação alguma sobre como reparar inconsistências nestes modelos. Nesta dissertação, mostramos que abordagens desenvolvidas para a atualização de modelos CTL inconsistentes não são capazes de lidar com todos os tipos de alterações em modelos. Introduzimos então o conceito de revisão de modelos: uma abordagem baseada em revisão de crenças para o reparo de modelos inconsistentes em um contexto estático. Relacionamos nossa proposta com trabalhos clássicos em revisão de crenças. Definimos um operador de revisão de modelos e mostramos que este obedece postulados de racionalidade clássico de revisão de crenças. Propomos um algoritmo de revisão com base no algoritmo utilizado pela abordagem de atualização de modelos. Discutimos sobre problemas e limites do algoritmo proposto, e mostramos que essa estratégia de adaptação não é uma solução apropriada.
Model checking is one of the most robust techniques in automated system verification. But, although this technique can handle complex verifications, model checking tools usually do not give any information on how to repair inconsistent system models. In this dissertation, we show that approaches developed for CTL model update cannot deal with all kinds of model changes. We introduce the concept of CTL model revision: an approach based on belief revision to handle system inconsistency in a static context. We relate our proposal to classical works on belief revision. We define an operator for model revision and we show that it obeys the classical rationality postulates of belief revision. We propose an algorithm for model revision based on the algorithm used by the model update approach. We discuss problems and limitations of our proposed algorithm and show that this strategy of adaptation is not an appropriate solution.

This dissertation study describes and presents an alternative method for delivering digital video, the MPEG-4 compression format, through the use of an analog cable network for direct viewing on television sets. The purpose of the study is to propose this technology as an alternative to the present cable network technologies in Uruguay and elsewhere in South America. Delivery standards that might be used over such a CATV network include DVB-C (digital video broadcast for cable), and IP (internet protocol) networks. The study compares the current technologies to determine which would be the best way to provide such delivery over CATV networks. It is the argument of this study that the delivery of MPEG-4 format streaming video is no different than the delivery of any type of digital signal: in order for the signal to be delivered, it must pass through a transmission medium such as fiber-optic cable, twisted-wire pair, coaxial cable, etc. The dissertation makes the case that MPEG-4 is a feasible alternative technology which should be considered for new CATV applications in this region. This study is from a technical point of view, when it comes to implementation there are other elements that should be taken into account.

Dissertação de Mestrado Integrado em Medicina Veterinária
O vírus da esgana (CDV) e o parvovírus (CPV) são dois vírus responsáveis por elevadas taxas de mortalidade nas populações caninas, que infetam cães não vacinados ou com protocolos vacinais incompletos. As vacinas têm desempenhado um papel fundamental na prevenção destas doenças no entanto, a vacinação não é um procedimento inócuo e deve ser realizado apenas com a frequência necessária para manter os cães imunizados. Por outro lado, são vários os fatores que podem originar falhas na imunização, sendo a neutralização do antigénio vacinal por anticorpos maternos a causa mais comum. Neste estudo foi avaliada a proteção humoral para o CDV e CPV a 13 cachorros durante a primovacinação (Grupo A e Grupo B) e, a 7 cães adultos, não vacinados há pelo menos 3 anos (Grupo C). O grupo A incluiu 5 cachorros com início do protocolo vacinal às 6 semanas e o grupo B contemplou 8 animais que iniciaram a primovacinação entre as 8 e as 12 semanas. De cada animal foram recolhidas três amostras de sangue, coincidentes com as consultas de vacinação, com 3 a 4 semanas de intervalo. A resposta humoral foi avaliada recorrendo a testes rápidos, baseados na técnica de ELISA indireta para deteção de anticorpos, que permitem avaliar de forma rápida, simples e económica a proteção ou suscetibilidade de cada animal. Verificámos que a resposta à vacinação para CDV foi mais precoce em comparação com a resposta para CPV. Relativamente ao CPV, 80% dos animais do grupo A, ainda se encontravam desprotegidos após a administração de duas doses da vacina. Já os animais do grupo B revelaram proteção humoral para CPV após duas administrações vacinais, sendo que quatro animais (50%) ficaram logo protegidos após a primeira dose. Nos cães adultos foi realizada apenas uma colheita de sangue que funcionou como um rastreio serológico para aferir a necessidade de revacinação. Os resultados obtidos sugerem que, durante a primovacinação, a resposta à vacinação é individual e depende sobretudo do título inicial de anticorpos maternos adquirido pelo neonato. A variabilidade encontrada reforça a necessidade da determinação dos níveis de imunidade humoral individuais. Deste modo, os kits de ELISA são uma ferramenta muito vantajosa, pois permitem avaliar a proteção a um custo relativamente reduzido. Estes testes deverão ser utilizados para validar a eficácia vacinal induzida pela primovacinação, auxiliando o médico veterinário a estabelecer protocolos vacinais individuais.
ABSTRACT - EVALUATION OF THE HUMORAL IMMUNE RESPONSE INDUCED BY VACCINATION FOR CANINE DISTEMPER AND PARVOVIRUS - Canine distemper virus (CDV) and parvovirus (CPV) are two viruses responsible for high mortality rates in the canine population that infect unvaccinated dogs and dogs with incomplete vaccination protocols. Vaccines continue to play a key role on the prevention of these diseases however vaccination is not an innocuous procedure and must be done only with the required frequency to keep dogs immunized. On the other hand, there are several factors that can lead to immunization failures, being the neutralization of the vaccine antigen by the maternal antibodies the main cause. In this study, the humoral protection for CDV and CPV was evaluated in 13 dogs during primary vaccination (Group A and Group B) and in 7 adult dogs that had not been vaccinated for at least 3 years (Group C). Group A included 5 dogs which started the vaccine protocol at 6 weeks and group B included 8 animals that started primary vaccination at 8 to 12 weeks. Three blood samples were collected from each animal, coincident with the vaccination visits, at 3 to 4 weeks apart. The humoral response was evaluated using rapid tests based on the indirect ELISA technique for antibody detection, which allow a rapid, simple and economical evaluation of the protection or susceptibility of each animal. We found that the response to CDV vaccination was precocious compared to the response to CPV. With regard to CPV, 80% of group A dogs were still unprotected after administration of two doses of the vaccine. In contrast, the dogs of group B showed humoral protection for CPV after two vaccination administrations, with four dogs (50%) being protected immediately after the first dose. In adult dogs, only one blood sample was taken for serological screening to estimate the need for revaccination. The results suggest that during primary vaccination the response to vaccination is individual and mostly depends on the initial titer of maternal antibodies acquired by the neonate. The variability found supports the need to measure individual humoral immunity levels. Thus the ELISA kits are a very helpful tool, because they allow the evaluation of the protection at a relatively reduced cost. These tests should be used to validate the vaccine efficacy induced by primary vaccination, helping the veterinarian to establish individual vaccination protocols.
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Rådande världsförhållanden riktar ökat fokus på frågor om hållbarhet. Den textila värdekedjans globala utspridning anstränger världens sociala som miljömässiga förhållanden. Därmed blir det allt viktigare för organisationer att engagera sig i ett arbete för att minimera effekterna av dess verksamhet. Företagens ansvarstagande- hållbarhetsaktiviteter benämns vanligen under Corporate Social Responsibility (CSR), begreppet är väletablerat och kritik av detta har givit upphov till nya benämningar. Creating Shared Value (CSV) är ett begrepp som fokuserar på att identifiera, utveckla och expandera kopplingen mellan ekonomisk och samhällelig tillväxt för att skapa simultant värde för likväl samhället som företagen. Syftet med denna studie var att, genom en fallstudie av ett svenskt modeföretag med utvecklat hållbarhetsarbete, undersöka de hållbarhetsaktiviteter som genomförs inom en textil värdekedjan ur ett CSV-perspektiv. Genom en kvalitativ forskningsdesign med deduktiv ansats studerades företaget och dess värdekedja i avsikt att besvara studiens frågeställning och uppnå dess syfte. Studiens empiriska material samlades in via semistrukturerade intervjuer med individer som representerade fallföretagets olika funktioner i värdekedjan. Materialet förklarades och tolkades utifrån det teoretiska ramverk som presenterats. Empirin och analysen visade att de genomförda aktiviteterna även kunde tolkas ur ett CSV-perspektiv under förutsättning att företagets tilläts tillämpa ett långsiktigt förhållningssätt och en identifiering av det delade värdet. Slutsatsen som drogs är att de undersökta hållbarhetsaktiviteterna genererar simultant värde för företag och samhället de verkar i. Aktiviteternas utfall och grad av värde varierade då vissa aktiviteter genererade ett direkt värde med påtagligt resultat för endera miljön eller samarbetspartners som på lång sikt skulle generera värde i konkurrensfördelar för fallföretaget medan andra hade en mer direkt effekt för båda inblandade parter. Generaliserbarheten av studiens resultat är begränsat på grund av den valda fallstudiemetoden men resultaten av denna studie anses likväl vara en intressant kontribution till forskningsdiskussionen om hållbarhetsaktiviteter.

This master thesis deals with analysis of structural variation of genome and with methods of its sequencing across all generations. Subsequently it contains a description of copy number variation and methods of its detection. The experimental part focuses on algorithm proposal for CNV detection according analysis and testing of uneven coverage in genome, variable representation of GC content and distance of sequence reads. Finally, the algorithm for detecting copy number variation is tested on genomic data of bacteria Klebsiella pneumoniae.

Copy number variation detection in prokaryotic organisms is currently receiving more and more attention, mainly due to the association of CNV with pathogenicity and antibiotic resistance in bacteria. The algorithm designed in this thesis uses peak detection in sequencing coverage to detect CNV segments. Read coverage is commonly obtained by mapping sequencing reads of one individual to an already known reference of another individual of the same species. However, two individuals will always differ in a certain number of genes, resulting in unmapped reads that are unnecessarily discarded. Therefore, this work assumes that the biological accuracy of CNV detection can be increased by using a new reference that is created from the same set of reads as the reads mapped to this reference. Sequencing reads of Klebsiella pneumoniae individuals are used to verify this assertion.

Mestrado em Engenharia Química
Os selantes butílicos, ou simplesmente mastiques, são produtos à base de borracha butílica que possibilitam a selagem, impedindo a passagem de fluidos através de superfícies e aberturas em diversos materiais. A facilidade de aplicação e a possibilidade de remoção após o seu uso, são duas vantagens que os distinguem de outros selantes, além de um rácio custo/benefício também bastante vantajoso. Estão disponíveis para venda, diluídos em solvente com aplicação a frio e isentos de solvente com aplicação a quente. Este projeto teve como objetivo desenvolver dois selantes de borracha butílica isentos de solvente (COV) para aplicação a frio: Selbutil-1 e Selbutil-2, tendo por base os produtos atualmente preparados pela empresa que contêm solvente (Sikabutyl-1 e Sikabutyl-2). Foi necessário reformular estes produtos no sentido de manter as suas características de desempenho ou, se possível, melhorá-las, diminuindo o impacto ambiental e torná-los menos perigosos para quem os manuseia. Desta forma recorreu-se a matérias-primas alternativas, seguindo-se o desenvolvimento de formulações e produção laboratorial. Efetuou-se um planeamento fatorial de experiências usando a metodologia de desenho de misturas para otimizar a percentagem de cargas, sílicas, plasticizantes e auxiliar de processo na formulação, sendo as variáveis resposta o escorrimento e o tempo de extrusão do selante. Os novos selantes Selbutil-1 e Selbutil-2 obtidos evidenciaram propriedades equivalentes aos de referência, nomeadamente, viscosidade, densidade, consistência e não possuem escorrimento. A única diferença foi a ausência de formação de pele, que se mostra estar associada à inclusão de solvente na preparação. As análises FTIR e os ângulos de contacto medidos sugerem que a composição à superfície do selante se modifica com a secagem do solvente possivelmente devido à migração preferencial de alguns constituintes da mistura como os plasticizantes.
The butyl sealants, or simply mastics, are butyl rubber based products that allow the sealing, preventing the passage of fluids through surfaces and openings in various materials. The ease of application and the possibility of removal after use, are two advantages that distinguish them from other sealants, as well as a quite advantageous cost / benefit ratio. They are available for sale, diluted in solvent for cold application and solvents free for heat application. This project aimed to develop two butyl rubber sealants free of solvent (VOC) for cold use: Selbutil-1 and Selbutil-2, which were based on two products currently produced by the company containing solvent (Sikabutyl-1 and Sikabutyl-2). It was necessary to reformulate these products to maintain its performance characteristics or if possible improve them, reducing the environmental impact and make them less dangerous for those who handle them. This way we used alternative raw materials, following the development of formulations and laboratory production. A factorial planning of experiences was developed using Mixture Design methodology to optimize the percentage of fillers, silicas, plasticizer and processing aid in the formulation, being response variables the outflow and the extruding time of the sealant. The new sealants obtained, Selbutil-1 and Selbutil-2, revealed equivalent properties to the references, in particular, viscosity, density, consistency and do not have outflow. The only difference was absence of skin formation, being a result of inclusion the solvent in the preparation. FTIR analyses and the measured angles of contact suggest that the composition at the surface of the sealant changes with the drying of the solvent, possibly due to preferential migration of some components of the mixture, as plasticizers.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia de Produção.
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Nos últimos anos, a economia mundial e a economia brasileira têm sofrido mudanças importantes. Fusões, aquisições e alianças estratégicas têm se multiplicado. Parte considerável destas mudanças relaciona-se com profundas alterações nos sistemas de valores de todos os segmentos industriais. A busca da competitividade relaciona-se cada vez mais com a busca do ótimo sistêmico além das fronteiras da empresa. Neste contexto, a administração logística ganha nova dimensão, envolvendo a integração de todas as atividades ao longo da cadeia de valores e do sistema de valores, das matérias-primas ao cliente final. A competitividade não é sustentada apenas na capacidade das organizações em criar novos produtos e serviços, e sim na oferta de valor aos seus clientes com o menor custo possível, especialmente nas indústrias em que a diferenciação de produtos pode não ser viável como a indústria do petróleo. No Brasil, como parte das mudanças no setor petróleo do país, o governo tomou várias medidas que visando eliminar os subsídios na distribuição e implantar a liberação de preços. Neste contexto - elevada concorrência, com o consumidor tendendo a optar pelo preço mais baixo - as grandes empresas do setor estão oferecendo uma variedade de serviços com o objetivo de manter os seus clientes. No sistema Petrobras a atividade de distribuição de derivados de petróleo é executada pela Petrobras Distribuidora. Entre os serviços oferecidos pela Petrobras Distribuidora, destaca-se o CTF - Controle Total de Frotas, voltado para o segmento de grandes empresas de transporte rodoviário. A proposta desse trabalho foi verificar a contribuição do CTF na agregação de valor para os clientes da Petrobrás Distribuidora. Inicialmente foi realizada uma revisão bibliográfica de modo a se conhecer o papel da logística na competitividade empresarial. Em seguida, através de um estudo de caso, buscou-se identificar no CTF os elementos logísticos que agregam valor aos clientes. Como conclusão do trabalho verificou-se o serviço logístico representado pelo CTF proporcionou uma agregação de valor ao criar condições para a melhoria da capacidade competitiva aos clientes do segmento de transporte rodoviário da Petrobras Distribuidora. In the last years the world and the Brazilian economy have been changing deeply. Fusions, acquisitions and strategic alliances are multiplying everywhere. Most of these changes are related with deep modifications in the value systems, affecting all industries. The search for competitiveness is more and more concerned with the search for the optimal systemic beyond organizational frontiers. Within this context, logistics management gains a new dimension, encompassing the integration of all activities related to the value system, from raw materials to the final customer. The competitiveness only is not supported in the capacity of the organizations in creating new products and services, and yes in it offers of value to its customers with the lesser possible cost, especially in the industries where the differentiation of products can not be viable as the oil industry. In Brazil, as part of the changes in the oil industry, the government took some measures that aiming at to eliminate the subsidies in the distribution and to implant the release of prices. In this context - raised competition, with the consumer tending to opt to the price lowest - the great companies of the sector are offering a variety of services with the objective to keep its customers. In the Petrobras system Petrobras Distribuidora executes the activity of distribution of oil derivatives. It enters the services offered for the Petrobras Distribuidora, is distinguished the CTF - Total Control of Fleets, come back toward the segment of great companies of road transport. The proposal of this work was to verify the contribution of the CTF in the aggregation of value for the customers of Petrobras Distribuidora. Initially a bibliographical revision was carried through in order to know the role of the logistic one in the enterprise competitiveness. After that, through a case study, one searched to identify in the CTF the logistic elements that add value to the customers. As conclusion of the work the logistic service represented by the CTF was verified provided to an aggregation of value when creating conditions for the improvement of the competitive capacity to the customers of the segment of road transport of the Petrobras Distribuidora.

Abstract Structure of membrane protiens can be determined by diffirent techniques.Electron Crystallography is one of the commonly used techniques to determine their structure in atomic or near atomic resolution.Due to the crystal disorder and poor CTF correction techniques the resolution obtained from this technique is not ideal.To push the resolution to the ideal, single particle refinement with local averaging for crystal disorder and improved CTF correction methodology for tilted data sets has been applied.With this approach applied to microsomal glutathione S-transferase 1(MGST1) membrane Protien data set comparable resolution with fewer data sets to the previous reconstruction has been achieved.

Additional Hydroxyl group on CT6 (3,4-dihydroxy-5,7-dimethoxyflavone), a flavone extracted from Chromolaena Tacotana potentially confers additional activity against pancreatic cancer as compared to CT7 (4-hydroxy-5,7-dimethoxyflavone) Parker Wade1, Miranda Green1, April Weaver1, Omri Coke1, Ruben D. Torrenegra2, and Victoria Palau1 1Department of Pharmaceutical Sciences, College of Pharmacy, East Tennessee State University, Johnson City, TN. 2Department of Chemistry, Universidad de Ciencias Aplicadas y Ambientales, Bogota, Colombia and Pancreatic cancer is one of the deadliest types of cancers, with a mortality rate of about 95%. This high mortality rate signifies there is a need for further research into finding treatment options for those affected by pancreatic cancer. Recent studies have found cytotoxic effects on cancerous cells elicited from compounds, such as flavones, in plants indigenous to Western South America, specifically Colombia. The flavones 3,4-dihydroxy-5,7-dimethoxyflavone (CT6) and 4-hydroxy-5,7-dimethoxyflavone (CT7) were isolated from Chromolaena Tacotana, member of the asteraceae family. The molecular structures of the flavones differ only by an additional hydroxyl group on CT6. Both of these compounds were tested on MIA PaCa2 and Panc28 pancreatic cancer cells at concentrations ranging from 5μM to 80μM. Cell viability after dosing of CT6 and CT7 was determined using MTT and spectrophotometry analysis. MIA PaCa2 is more poorly differentiated than Panc28. CT6 conferred greater activity on both cell lines compared to CT7. Percent cell viability of the Panc28 cell line reached a low of 35.55% (p=0.0001) with CT6, compared to 84.25% (p=0.0275) with CT7. Percent cell viability of the MIA PaCa2 cell line reached a low of 46.72% (p=0.000001)with CT6. However, CT7 showed no significant difference, with percent cell viability reaching 103.73% (p=0.5605) when compared to the control for this cell line. While CT6 exerted cytotoxic activity on both Panc28 and MIA PaCa2, CT6 had significantly more cytotoxic activity on Panc28, which could be related to the greater differentiation status of this cell line. More in depth studies will need to be conducted to determine the exact reasons for greater activity of CT6 on Panc28 cells. This could be due to the compound’s target, mitochondrial activity of the cell lines, and the minor structural differences between the two compounds.