A cultura do tomateiro (Solanum lycopersicum L.) é importante mundialmente devido ao alto consumo de seus frutos. Nos últimos anos surgiram nesta cultura no Brasil alguns vírus emergentes com altas taxas de disseminação, como begomovírus e crinivírus, transmitidos pela Bemisia tabaci biótipo B, que podem causar danos à produção do tomateiro. A espécie de begomovírus atualmente mais encontrada no Brasil, em plantios de tomateiro, é o Tomato severe rugose virus (ToSRV). De 2002 a 2004, pesquisadores relataram incidências desse vírus em mais da metade das amostras com sintomas de geminiviroses coletadas em vários estados brasileiros e sua presença continua sendo verificada frequentemente. No ano de 2006, um crinivírus, o Tomato chlorosis virus (ToCV), foi relatado no Brasil, infectando plantas de tomate no Estado de São Paulo e atualmente encontra-se presente em diveros estados brasileiros. Os objetivos desse trabalho foram: determinar os períodos mínimos de acesso à aquisição e à inoculação do ToSRV e do ToCV pela B. tabaci biótipo B; identificar o período de retenção do ToSRV no inseto e a interação do ToSRV e do ToCV na aquisição e na transmissão por esse aleirodídeo. Também foi avaliada a eficiência do inseticida cloridrato de cartape no controle da disseminação primária e secundária do ToSRV pela B. tabaci biótipo B em tomateiros em gaiolas em casa de vegetação. Finalmente avaliou-se a eficiência do aleirodídeo Trialeurodes vaporariorum na transmissão de um isolado brasileiro do ToCV. Os períodos mínimos de acesso à aquisição e à inoculação de ambos os vírus pela B. tabaci biótipo B foram de cinco minutos. O tempo de retenção do ToSRV em B. tabaci biótipo B foi de 25 dias. A eficiência de um único adulto de B. tabaci na transmissão simultânea do ToSRV e do ToCV para tomateiros foi de 44,7%, similar àquela da transmissão isolada do ToRSV (47,4%) e do ToCV (44,7%). A eficiência de T. vaporariorum na transmissão do ToCV foi inferior à da B. tabaci biótipo B. Usando 40 insetos por vaso com duas plantas as eficiências de transmissão foram 57,7% e 100%, respectivamente. O inseticida cloridrato de cartape reduziu a infecção secundária do ToSRV pela B. tabaci biótipo B, mas não foi eficiente para reduzir a infecção primária em tomateiros. / Tomato (Solanum lycopersicum) is one of the leading vegetables grown and consumed in Brazil and in the world, after potato. The importance of tomato is related to its high consumption worldwide and also its nutritive value. Presently the most important virus diseases responsible for yield losses on tomato crops in Brazil are those caused by begomovirus and crinivirus, both transmitted by Bemisia tabaci biotype B. At the moment the prevalent species of begomovirus is Tomato severe rugose virus (ToSRV). From 2002 to 2004, researchers reported incidence of this virus in more than half of the symptomatic tomato samples collected in several Brazilian states. In 2006, a crinivirus, Tomato chlorosis virus (ToCV), was reported for the first time in Brazil, infecting tomato plants in the State of São Paulo and at present the virus occurs in several Brazilian states. The objectives of this study were to determine the minimum acquisition and inoculation access periods of ToSRV and ToCV by B. tabaci biotype B; identify the retention period of ToSRV in the insect; and the interaction of ToSRV and ToCV on the transmission by this aleyrodidae. It was also evaluated the effectiveness of the insecticide cartap hydrochloride in controlling the primary and secondary spread of ToSRV by B. tabaci biotype B on tomato plants in a greenhouse. Finally, it was evaluated the efficiency of Trialeurodes vaporariorum in the transmission of a Brazilian isolate of ToCV. The minimum acquisition and inoculation access periods for both viruses by B. tabaci biotype B were five minutes. The maximum retention time of ToSRV in B. tabaci biotype B was 25 days. The efficiency of a single adult of B. tabaci to simultaneously transmit ToSRV and ToCV to tomato plants was 44.7%, similar to the transmission of ToRSV (47.4%), and ToCV (44.7%) separately. T. vaporariorum was less efficient than B. tabaci on the transmission of ToCV. Using 40 insects per pot with two plants, transmission efficiencies were 57.7% and 100%, respectively. The insecticide cartap hydrochloride reduced secondary infection of ToSRV transmitted by B. tabaci biotype B, but was not effective in reducing the primary infection in tomato.

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.

The Tobacco mosaic virus (TMV) is a simple and frequently used model virus which has been studied already more over than 130 years. Due to the intensive study of this virus the details of its infectious cycle, genomic information and also the structure of the created viral particle as well as the mechanism of its creation are known today. The process of encapsidation (viral particle formation) is sufficiently described in the in vitro conditions. In the in vitro conditions the origin of assembly (OAS) was also described. The OAS was identified in the coding sequence of the gene for the movement protein (MP). The importance of replication centers (replication factories) has also been supposed. The aim of the diploma thesis was to study the specificity of the interaction of RNA and coat protein in the process of the particle assembly taking place directly inside the plants. The experiments were performed to verify the necessity of presence of OAS sequence in process of initiation of viral encapsidation. The effect of the cell compartmentation on this process has also been studied. Based on several viral systems (the Tobacco mosaic virus, the Potato virus X, the Bean yellow dwarf virus and Cowpea mosaic virus) gene constructs were created. These constructs enables to study this idea at the molecular...

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No estado do Espírito Santo (ES), uma das principais áreas produtoras de mamão do país, a eliminação sistemática de plantas doentes tem sido aplicada desde a década de 1980 para o controle do mosaico do mamoeiro (Papaya ringspot virus - type P; PRSV-P). O uso permanente dessa prática nos últimos 25 anos levou a uma aparente seleção e predominância de isolados fracos do vírus. Os objetivos deste trabalho foram: investigar a prevalência desses isolados fracos, bem como a estabilidade e o efeito protetor contra isolados severos do vírus; estudar a infecção natural de abobrinha de moita (Cucurbita pepo cv. Caserta) e abóbora moranga (C. maxima cv. Exposição) com o PRSV-P quando plantadas ao lado de mamoeiros infectados e caracterizar um isolado do Alfalfa mosaic virus (AMV) em infecção natural em mamoeiro. A detecção de possíveis isolados fracos do vírus foi realizada por PTAELISA, microscopia eletrônica e RT-PCR. Todos os isolados também foram inoculados mecanicamente em mamoeiro cv. Golden para avaliação de sintomas. Sequências de nucleotídeos e de aminoácidos deduzidos do gene da proteína capsidial de alguns isolados fracos mostraram identidades superiores a 89% e 90%, respectivamente, com isolados do PRSV-P. De 119 amostras de mamoeiros analisadas, 86 estavam infectadas com o PRSV-P, mas somente 75 induziram sintomas fracos em mamoeiros. Quatro isolados fracos foram selecionados ao acaso para estudos de estabilidade, e de proteção em casa de vegetação. Apenas dois isolados fracos induziram sintomas estáveis em mamoeiros até a oitava transferência. A proteção total só foi obtida com plantas premunizadas com dois isolados fracos e desafiados com o isolado PRSV-PES. Plantas de mamoeiros cv. Golden premunizadas com vários isolados fracos do PRSV-P foram expostas em condições de campo na ESALQ/USP, Piracicaba, SP, em dois experimentos independentes. Poucas plantas permaneceram com sintomas fracos de mosaico até o final dos experimentos. Uma terceira exposição foi realizada em Linhares, ES, com mamoeiros cvs. Sunrise Solo e Golden premunizados com oito isolados fracos, coletados nos experimentos em campo na ESALQ/USP. Apenas uma planta premunizada com um isolado fraco permaneceu com sintomas leves da doença até a última avaliação. Tentativas de detectaçao de infecções naturais de cucurbitáceas com o PRSV-P foram realizados em dois plantios de abobrinha de moita e dois de abóbora moranga, na ESALQ/USP, Piracicaba, SP. A detecção do vírus foi feita por meio da inoculação de extratos foliares das cucurbitáceas em mamoeiros cv. Golden. Os mamoerios foram avaliados por meio de sintomas, PTA-ELISA e RT-PCR. Nenhuma planta de mamoeiro inoculada com extratos foliares das duas cucurbitáceas exibiu sintomas de mosaico, embora o gene ci, mas não o cp, tenha sido detectado em uma amostra de folhas de mamoeiro, indicando que ao menos uma planta de abobrinha de moita estava infectada. Finalmente, no decorrer dos ensaios de campo na ESALQ/USP, constatou-se uma planta de mamoeiro apresentando sintomas severos de mosaico amarelo, deformação foliar e necrose sistêmica, diferente daqueles induzidos pelo PRSV-P. Análises biológicas, sorológica e moleculares confirmaram tratar-se do AMV. Este é o primeiro relato de infecção natural de mamoeiro com esse vírus. / Papaya ringspot virus type P (PRSV-P) causes the major disease in Brazilian papaya orchards that result in significant yield losses. In Espírito Santo state systematic rouging of infected plants has been applied since early 1980s for the control of this disease. Its permanent use over the last 25 years has lead to an apparent selection and predominance of mild strains throughout papaya orchards. The objectives of this work were to investigate the prevalence of mild isolates, as well the stability and protective effect against severe isolates of the virus; The aim of this work was to study the natural infecction of zucchini squash (Cucurbita pepo cv. Caserta) and pumpkin (C. maxima cv. Exposição) grown near to papaya trees infected with PRSV-P and characterize an isolate of Alfalfa mosaic virus (AMV) in natural infection in papaya. The detection of possible mild isolates of the virus was performed by PTA-ELISA, electron microscopy and RT-PCR. All isolates were inoculated mechanically in papaya cv. Golden for symptoms evaluation. Nucleotides and deduced amino acids sequences of the coat protein gene of some mild isolates showed identities above 89% and 90%, respectively, with isolates of PRSV-P. Of 119 samples from papaya plants analyzed, 86 were infected with PRSV-P and 75 induced mild symptoms on papaya. Four mild isolates were randomly selected for stability and protection studies under greenhouse. Only two isolates induced mild symptoms on papaya and remained stables until the eighth transference. Full protection was obtained with preimmunized plants with two mild isolates and challenged with the isolate PRSV-P-ES. Plants of papaya cv. Golden preimmunized with several mild isolates of PRSV-P were exposed under field conditions at ESALQ/USP, Piracicaba,SP, in two independent experiments. Few plants remained with mild mosaic symptoms at the end of the experiments. A third field exposition was held in Linhares,ES, with papaya cvs. Golden and Sunrise Solo preimmunized with eight mild isolates, collected in field experiments at ESALQ/USP. Only one plant preimmunized with a mild isolate remained with mild symptoms of the disease until the last evaluation. Attempts of detection of natural infections of cucurbits with PRSV-P were carried out in two plantations of zucchini squash and pumpkin at ESALQ/USP, Piracicaba,SP. The detection of the virus was made by inoculation of leaf extracts of cucurbits in papaya cv. Golden. The papaya plants were assessed by symptoms, PTAELISA and RT-PCR. None of papaya plants exhibited symptoms of mosaic, while the ci gene, but not the cp, was detected in a sample of leaves of papaya, indicating that at least one clump of zucchini squash plant was infected. Finally, during the field test at ESALQ/USP, a papaya plant was found showing severe symptoms severe yellow leaf mosaic, leaf distortion and systemic necrosis, different from those induced by PRSV-P. Biological, serological and molecular tests confirmed the infection with AMV. This is the first report of natural infection of papaya with this virus.

No estado do Espírito Santo (ES), uma das principais áreas produtoras de mamão do país, a eliminação sistemática de plantas doentes tem sido aplicada desde a década de 1980 para o controle do mosaico do mamoeiro (Papaya ringspot virus - type P; PRSV-P). O uso permanente dessa prática nos últimos 25 anos levou a uma aparente seleção e predominância de isolados fracos do vírus. Os objetivos deste trabalho foram: investigar a prevalência desses isolados fracos, bem como a estabilidade e o efeito protetor contra isolados severos do vírus; estudar a infecção natural de abobrinha de moita (Cucurbita pepo cv. Caserta) e abóbora moranga (C. maxima cv. Exposição) com o PRSV-P quando plantadas ao lado de mamoeiros infectados e caracterizar um isolado do Alfalfa mosaic virus (AMV) em infecção natural em mamoeiro. A detecção de possíveis isolados fracos do vírus foi realizada por PTAELISA, microscopia eletrônica e RT-PCR. Todos os isolados também foram inoculados mecanicamente em mamoeiro cv. Golden para avaliação de sintomas. Sequências de nucleotídeos e de aminoácidos deduzidos do gene da proteína capsidial de alguns isolados fracos mostraram identidades superiores a 89% e 90%, respectivamente, com isolados do PRSV-P. De 119 amostras de mamoeiros analisadas, 86 estavam infectadas com o PRSV-P, mas somente 75 induziram sintomas fracos em mamoeiros. Quatro isolados fracos foram selecionados ao acaso para estudos de estabilidade, e de proteção em casa de vegetação. Apenas dois isolados fracos induziram sintomas estáveis em mamoeiros até a oitava transferência. A proteção total só foi obtida com plantas premunizadas com dois isolados fracos e desafiados com o isolado PRSV-PES. Plantas de mamoeiros cv. Golden premunizadas com vários isolados fracos do PRSV-P foram expostas em condições de campo na ESALQ/USP, Piracicaba, SP, em dois experimentos independentes. Poucas plantas permaneceram com sintomas fracos de mosaico até o final dos experimentos. Uma terceira exposição foi realizada em Linhares, ES, com mamoeiros cvs. Sunrise Solo e Golden premunizados com oito isolados fracos, coletados nos experimentos em campo na ESALQ/USP. Apenas uma planta premunizada com um isolado fraco permaneceu com sintomas leves da doença até a última avaliação. Tentativas de detectaçao de infecções naturais de cucurbitáceas com o PRSV-P foram realizados em dois plantios de abobrinha de moita e dois de abóbora moranga, na ESALQ/USP, Piracicaba, SP. A detecção do vírus foi feita por meio da inoculação de extratos foliares das cucurbitáceas em mamoeiros cv. Golden. Os mamoerios foram avaliados por meio de sintomas, PTA-ELISA e RT-PCR. Nenhuma planta de mamoeiro inoculada com extratos foliares das duas cucurbitáceas exibiu sintomas de mosaico, embora o gene ci, mas não o cp, tenha sido detectado em uma amostra de folhas de mamoeiro, indicando que ao menos uma planta de abobrinha de moita estava infectada. Finalmente, no decorrer dos ensaios de campo na ESALQ/USP, constatou-se uma planta de mamoeiro apresentando sintomas severos de mosaico amarelo, deformação foliar e necrose sistêmica, diferente daqueles induzidos pelo PRSV-P. Análises biológicas, sorológica e moleculares confirmaram tratar-se do AMV. Este é o primeiro relato de infecção natural de mamoeiro com esse vírus. / Papaya ringspot virus type P (PRSV-P) causes the major disease in Brazilian papaya orchards that result in significant yield losses. In Espírito Santo state systematic rouging of infected plants has been applied since early 1980s for the control of this disease. Its permanent use over the last 25 years has lead to an apparent selection and predominance of mild strains throughout papaya orchards. The objectives of this work were to investigate the prevalence of mild isolates, as well the stability and protective effect against severe isolates of the virus; The aim of this work was to study the natural infecction of zucchini squash (Cucurbita pepo cv. Caserta) and pumpkin (C. maxima cv. Exposição) grown near to papaya trees infected with PRSV-P and characterize an isolate of Alfalfa mosaic virus (AMV) in natural infection in papaya. The detection of possible mild isolates of the virus was performed by PTA-ELISA, electron microscopy and RT-PCR. All isolates were inoculated mechanically in papaya cv. Golden for symptoms evaluation. Nucleotides and deduced amino acids sequences of the coat protein gene of some mild isolates showed identities above 89% and 90%, respectively, with isolates of PRSV-P. Of 119 samples from papaya plants analyzed, 86 were infected with PRSV-P and 75 induced mild symptoms on papaya. Four mild isolates were randomly selected for stability and protection studies under greenhouse. Only two isolates induced mild symptoms on papaya and remained stables until the eighth transference. Full protection was obtained with preimmunized plants with two mild isolates and challenged with the isolate PRSV-P-ES. Plants of papaya cv. Golden preimmunized with several mild isolates of PRSV-P were exposed under field conditions at ESALQ/USP, Piracicaba,SP, in two independent experiments. Few plants remained with mild mosaic symptoms at the end of the experiments. A third field exposition was held in Linhares,ES, with papaya cvs. Golden and Sunrise Solo preimmunized with eight mild isolates, collected in field experiments at ESALQ/USP. Only one plant preimmunized with a mild isolate remained with mild symptoms of the disease until the last evaluation. Attempts of detection of natural infections of cucurbits with PRSV-P were carried out in two plantations of zucchini squash and pumpkin at ESALQ/USP, Piracicaba,SP. The detection of the virus was made by inoculation of leaf extracts of cucurbits in papaya cv. Golden. The papaya plants were assessed by symptoms, PTAELISA and RT-PCR. None of papaya plants exhibited symptoms of mosaic, while the ci gene, but not the cp, was detected in a sample of leaves of papaya, indicating that at least one clump of zucchini squash plant was infected. Finally, during the field test at ESALQ/USP, a papaya plant was found showing severe symptoms severe yellow leaf mosaic, leaf distortion and systemic necrosis, different from those induced by PRSV-P. Biological, serological and molecular tests confirmed the infection with AMV. This is the first report of natural infection of papaya with this virus.

Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this study are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.

Chapter 3 Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this chapter, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. Further NSm was shown to be phosphorylated by N. benthamiana and tomato crude sap as observed in other movement proteins. Chapter 4 This chapter deals with localization of NSm to PD and identification of domain involved in localization. For this purpose NSm and its mutants were cloned in pEAQ:GFP vector and transiently expressed in N. benthamiana by infiltration of transformed Agrobacteria. The GFP tagged NSm was visualized by confocal microscopy. The results demonstrated that NSm forms punctate structures and localizes to PD as confirmed by colocalization of mCherry: PDLP1a, a PD marker which resides in PD, with GFP:NSm. To find out the domain involved in PD localization, sequential deletion mutants were made. It was found that C-terminal domain is involved in PD localization. On the other hand, N-terminal unfolded region was dispensable for PD localization. This is the first report of a coiled coil domain shown to be involved in PD localization. It has also been demonstrated that GBNV NSm interacts with NP. Further, membrane floatation assay carried in presence of NP suggested that interaction of NSm and NP affected membrane association of NSm. These results were further confirmed by localization studies of NSm in presence of NP. It was found that there was considerable relocalization of both NSm and NP. NSm was observed to be present in cytoplasm as well as on the membrane. At the same time, NP was observed on membrane apart from being present in the cytoplasm. When N-terminal 50 amino acids (unfolded) region of NSm was deleted and colocalization studies were carried out, it was found that NSm and NP do not colocalize, suggesting that NSm interacts with NP via the unfolded region and helps in the relocalization of NP to the membrane. Chapter 5 This chapter deals with the pathway of targeting NSm to PD. To decipher the pathway, followed by NSm, an inhibitor of endomembrane or vesicle mediated transport, Brefeldin A (BFA) was used. When GFP-NSm was expressed it was observed to form punctate structure at PD as before. Upon treatment with BFA, green islands were observed in the cytoplasm suggesting that ER was involved in targeting NSm to PD. Similarly, LatB, inhibitor of actin mediated targeting of protein to membrane, also abrogated the localization of NSm to PD. In order to further understand the role of ER in targeting NSm to PD, an ER marker, ER-GFP (GFP fused to HDEL peptide that directs it to ER) was coexpressed with GBNV NSm fused to mCherry. It was observed that NSm colocalizes with ER-GFP as yellow puncta on PD. The puncta appeared as patches and the whole ER-network was converted to vesicles. This was further confirmed by coexpressing ER-GFP with NSm without any tag. The green fluorescent vesicles were observed preferentially near cell membrane. To delineate the region of NSm involved in vesicle formation, point mutants and deletion mutants of NSm were generated without the tag and coexpressed with ER-GFP. When N-terminal 203 amino acids were deleted, NSm was able to transform ER membranes to vesicles suggesting that these residues are dispensable for vesicle formation. Interestingly, the deletion of coiled coil domain leads to cytosolic location of NSm. Furthermore, the C-terminal coiled coil domain when expressed alone was capable of inducing vesicle formation. This is the first report of involvement of such a domain in ER membrane association and vesicle formation.

[ES] Hoy en día, los virus de las plantas no sólo se perciben como patógenos, sino que también son capaces de establecer una asociación beneficiosa con sus huéspedes y colaborar desde un punto de vista biotecnológico. El virus, como vector, puede ser una herramienta para introducir genes heterólogos en la planta, que se procesarán junto con la información viral y producirán valiosas proteínas recombinantes, metabolitos o nanopartículas. Los vectores virales también son capaces de interferir con la maquinaria de silenciamiento de la planta dando prioridad a los genes virales. Los potyvirus son virus de ARN de plantas, que codifican principalmente una poliproteína con unas diez proteínas maduras con diferentes funciones, de las cuales las responsables de la supresión del silenciamiento son la proteína componente auxiliar (HC-Pro) y la proteína viral ligada al genoma (VPg). En la primera parte de este trabajo, utilizamos un aislado suave del virus del mosaico de la sandía (WMV; género Potyvirus, familia Potyviridae) para construir un vector para el silenciamiento génico inducido por virus (VIGS) en plantas de melón. Utilizando este virus como vector, expresamos un fragmento de ARNm de la Fitoeno desaturasa (PDS) de melón en las modalidades sentido, antisentido y horquilla para investigar el efecto de la construcción viral en el silenciamiento génico. Los resultados mostraron una expresión estable del fragmento de secuencia insertado en la planta en ambas orientaciones, sentido y antisentido, mientras que en la modalidad de horquilla el inserto se perdió pronto. Sin embargo, las tres construcciones indujeron el silenciamiento del gen PDS endógeno. Se confirmó la utilidad del WMV para el análisis genético inverso en melón expresando un fragmento de la subunidad I de la quelatasa de magnesio (CHLI). En general, nuestros resultados apoyaron que el vector WMV es útil para aplicar la tecnología VIGS en melón y, posiblemente, en otras cucurbitáceas. En la segunda parte de este trabajo, con el objetivo de fortificar las frutas comestibles con metabolitos promotores de la salud, se utilizó un vector de ARN viral derivado del virus del mosaico amarillo del calabacín (ZYMV; género Potyvirus, familia Potyviridae) para expresar una fitoeno sintasa bacteriana (crtB) en los frutos del calabacín (Cucurbita pepo L.). Esta enzima cataliza el primer paso de la biosíntesis de carotenoides. La expresión de la crtB mediada por el ZYMV dio lugar a la sobreacumulación de una serie de metabolitos de carotenoides y tocoferoles, concretamente α- y β-caroteno (provitamina A), luteína y fitoeno, así como α- y γ-tocoferol (vitamina E), tanto en la piel como en la pulpa del calabacín. Este resultado ilustra cómo se pueden enriquecer metabólicamente las frutas comestibles utilizando vectores virales sin modificar el genoma de la planta. / [CA] A dia de hui, els virus de les plantes no sols es perceben com a patògens, sinó que també són capaços d'establir una associació beneficiosa amb els seus hostes i col·laborar des d'un punt de vista biotecnològic. El virus, com a vector, pot ser una eina per a introduir gens heteròlegs en la planta, que es processaran juntament amb la informació viral i produiran valuoses proteïnes recombinants, metabòlits o nanopartícules. Els vectors virals també són capaços d'interferir amb la maquinària de silenciament de la planta donant prioritat als gens virals. Els potyvirus són virus d'ARN de plantes, que codifiquen principalment una poliproteïna d'unes deu proteïnes madures amb diferents funcions, de les quals les responsables de la supressió del silenciament són la proteïna component auxiliar (HC-Pro) i la proteïna viral lligada al genoma (VPg). En la primera part d'aquest treball, utilitzem un aïllat suau del virus del mosaic del meló d'Alger (WMV; gènere Potyvirus, família Potyviridae) per a construir un vector per al silenciament gènic induït per virus (VIGS) en plantes de meló. Utilitzant aquest virus com a vector, expressem un fragment d'ARNm de la Fitoeno desaturasa (PDS) del meló en les modalitats de sentit, antisentit i forqueta per a investigar l'efecte de la construcció viral en el silenciament gènic. Els resultats van mostrar una expressió estable del fragment de seqüència inserit en la planta en totes dues orientacions, sentit i antisentit, mentre que en la modalitat de forqueta l'inserit es va perdre prompte. No obstant això, les tres construccions van induir el silenciament del gen PDS endogen. Es va confirmar la utilitat de la WMV per a l'anàlisi genètica inversa en meló expressant un fragment de la Subunitat I de la quelatasa de magnesi (CHLI). En general, els nostres resultats van secundar que el vector WMV és útil per a aplicar la tecnologia VIGS en meló i, possiblement, en altres cucurbitáceas. En la segona part d'aquest treball, amb l'objectiu de fortificar les fruites comestibles amb metabòlits promotors de la salut, es va utilitzar un vector d'ARN viral derivat del virus del mosaic groc de la carabasseta (ZYMV; gènere Potyvirus, família Potyviridae) per a expressar una fitoeno sintasa bacteriana (crtB) en els fruits de la carabasseta (Cucurbita pepo L.). Aquest enzim catalitza el primer pas compromés de la biosíntesi de carotenoides. L'expressió de la crtB mediada pel ZYMV va donar lloc a la sobreacumulación d'una sèrie de metabòlits de carotenoides i tocoferoles, concretament α- i β caroté (provitamina A), luteïna i fitoeno, així com α- i γ--tocoferol (vitamina E), tant en la pell com en la polpa de la carabasseta. Aquest resultat il·lustra com es poden enriquir metabólicamente les fruites comestibles utilitzant vectors virals sense modificar el genoma de la planta. / [EN] Nowadays, plant viruses are not only perceived as pathogens, but also able to build a beneficial partnership with their hosts and co-work together from a biotechnological view. The virus, as a vector, can be a tool to introduce heterologous genes into the plant, which will process together with the viral information and produce valuable recombinant proteins, metabolites or nanoparticles. Viral vectors are also able to interfere with plant silencing machinery giving priority to the viral genes. Potyviruses are plant RNA viruses, mainly encoding a polyprotein of about ten mature proteins with different functions, from which the responsible of silencing suppression are helper-component proteinase (HC-Pro) and the viral protein genome-linked (VPg). In the first part of this work, we used a mild isolate of Watermelon mosaic virus (WMV; genus Potyvirus, family Potyviridae) to build a vector for virus induced gene silencing (VIGS) in melon plants. Using this virus as a vector, we expressed a fragment of melon Phytoene desaturase (PDS) mRNA in sense, antisense, and hairpin modalities to investigate the effect of the viral construct on gene silencing. The results showed a stable expression of the inserted sequence fragment in the plant in both sense and antisense orientations, whereas in the hairpin modality the insert was soon lost. Yet, all three constructs induced silencing of the endogenous PDS gene. The usefulness of the WMV for reverse genetic analysis in melon was confirmed expressing a fragment of Magnesium chelatase subunit I (CHLI). Overall, our results supported that the WMV vector is useful to apply the VIGS technology in melon and, possibly, other cucurbits. In the second part of this work, with the aim to fortify edible fruits with health promoting metabolites, a viral RNA vector derived from Zucchini yellow mosaic virus (ZYMV; genus Potyvirus, family Potyviridae) was used to express a bacterial phytoene synthase (crtB) in zucchini (Cucurbita pepo L.) fruits. This enzyme catalyzes the first committed step of carotenoid biosynthesis. The crtB expression mediated by ZYMV resulted in the overaccumulation of a range of carotenoids and tocopherols metabolites, namely α- and ß-carotene (pro-vitamin A), lutein and phytoene, as well as α- and γ-tocopherol (vitamin E), in both zucchini rind and flesh. This result illustrates how edible fruits can be metabolically fortified using viral vectors without plant genome modification. / This research was supported by grants BIO2017-83184-R, AGL2017-85563-C2-1-R and RTA2017-00061-C03-03 from Ministerio de Ciencia e Innovación (Spain), through Agencia Estatal de Investigación (cofinanced European Regional Development Fund). / Houhou, F. (2021). Biotechnological Application of Potyvirus-Based Vectors in Cucurbit Plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/177644 / TESIS