Modern agriculture is dependent on both global supply chains and crop monocultures. These features aid the evolution and spread of novel plant pathogens. Limited genetic diversity in commercial crop lines can result in widespread susceptibility to emerging pathogens. Pathogen resistance may be developed through conventional breeding approaches, or a number of transgenic strategies. This thesis focuses on the characterisation of an emerging maize disease, Maize lethal necrosis (MLN), and engineering resistant maize lines using an artificial microRNA (amiRNA) approach. MLN is a synergistic viral disease caused by the interaction of Maize chlorotic mottle virus (MCMV) with any maize-infecting member of the potyviridae. I used next-generation RNA sequencing to characterise the MLN outbreak in East Africa, discovering that local and Chinese strains of the potyvirus Sugarcane mosaic virus (SCMV) typically coinfect with MCMV. A first global MCMV phylogeny was constructed using these samples combined with new Sanger sequencing of samples in Ecuador and Hawaii. The phylogeny supported previous hypotheses of a link between the Chinese and African outbreaks, and suggested a novel link between the Hawaiian and Ecuadorian outbreaks. The SCMV sequences generated demonstrated strong evidence of extensive recombination, in line with previous reports on SCMV and potyviruses. These data also produced first reports of a number of RNA viruses in East Africa, and five novel viral-like sequences, with their presence confirmed by RT-PCR. RNA silencing is an important component of the plant immune response to viral infection. amiRNAs can be used to generate specific and effective viral resistance through Watson- Crick base pairing between the amiRNA and the (RNA) viral genome. Previous amiRNA approaches have targeted invariable genomic regions using consensus sequences. However, the high mutation rate of RNA viruses means single cells contain a variety of mutant genomes, collectively called a quasispecies. To deter the evolution of resistance breaking I devised a novel strategy to include intra-sample variation from NGS data in amiRNA design, and constructs, each containing five of these amiRNAs, were transformed into tropical maize lines. RNA silencing may be hampered by the expression of viral suppressors of silencing (VSRs). Local VSR assays demonstrated that there are no local VSRs in the MCMV genome, while systemic VSR assays showed a possible systemic VSR role for the unique P32 protein, and an interesting link between photoperiod and systemic silencing more generally.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, 2007. / Submitted by Ruthléa Nascimento (ruthleanascimento@bce.unb.br) on 2015-10-05T16:20:07Z No. of bitstreams: 1 Dissert_Anelise Orilio.pdf: 2146597 bytes, checksum: 218ace7315aad0f1ee3fc2b4fd23b756 (MD5) / Approved for entry into archive by Ruthléa Nascimento(ruthleanascimento@bce.unb.br) on 2015-10-05T16:20:31Z (GMT) No. of bitstreams: 1 Dissert_Anelise Orilio.pdf: 2146597 bytes, checksum: 218ace7315aad0f1ee3fc2b4fd23b756 (MD5) / Made available in DSpace on 2015-10-05T16:20:31Z (GMT). No. of bitstreams: 1 Dissert_Anelise Orilio.pdf: 2146597 bytes, checksum: 218ace7315aad0f1ee3fc2b4fd23b756 (MD5) / A mandioquinha-salsa é uma planta propagada vegetativamente e durante o seu cultivo sucessivo pode ocorrer o acúmulo de patógenos de infecção sistêmica como vírus, resultando em decréscimo da produção. No Brasil, é comum observar plantas de mandioquinha-salsa apresentando sintomas de mosaico, mosqueamento e deformação foliar, semelhantes a infecção por vírus. Evidências de ocorrência de vírus infectando esta cultura já foram relatadas, porém nenhum estudo de identificação e caracterização havia sido realizado. O objetivo do presente trabalho foi caracterizar biológica, sorológica e molecularmente um vírus isolado de mandioquinha-salsa e desenvolver técnicas para a sua detecção. Um isolado de vírus foi obtido a partir de mandioquinha-salsa coletada para estudo de micropropagação, denominado isolado C17, e que foi selecionado para a caracterização. Este vírus causa em Nicotiana benthamiana, hospedeiro usado para manutenção, sintoma de deformação foliar, bolhosidades e mosaico. O isolamento biológico foi realizado a partir do extrato da planta original de mandioquinha-salsa infectada em Chenopodium quinoa. Este isolado apresentou círculo de hospedeiros restrito e foi transmitido por afídeos de maneira não persistente. Partículas virais foram purificadas e análise ao microscópio eletrônico revelou partículas alongadas e flexuosas, típicas de espécies de Potyvirus. Em gel de poliacrilamida, a capa protéica apresentou uma proteína com peso molecular de aproximadamente 33 kDa, que em teste de Western blotting foi reconhecida especificamente pelo anti-soro policlonal produzido contra a proteína da capa do isolado C17. O anti-soro produzido em coelho a partir de partículas purificadas apresentou alta especificidade e sensibilidade. Amostras de campo do banco ativo de germoplasma de mandioquinha-salsa da Embrapa Hortaliças foram avaliadas por ELISA. A detecção foi positiva em 93 % das amostras, demonstrando a grande aplicabilidade do antisoro em testes diagnósticos de amostras do campo. A porção 3´ terminal do genoma do vírus (1,7 kb) foi clonada por RT- PCR e sequenciada em sequenciador automático. A análise da seqüência nucleotídica do fragmento clonado revelou uma fase aberta de leitura incompleta (sem o códon de iniciação) que codifica uma parte da proteína NIb (polimerase viral) e capa protéica (CP), seguida de uma região 3´ não traduzida (3’ UTR). A sequência nucleotídica da CP e da 3' UTR apresentou baixa porcentagem de identidade com seqüências de outros potyvírus depositados em bancos de dados públicos. A maior porcentagem de identidade nucleotídica da CP foi de 63 % para Araujia mosaic virus e da seqüência de aminoácidos foi de 60 % para Narcissus late season yellows virus (NLSYV). A análise filogenética confirmou o agrupamento do C17 com o NLSYV e sugeriu que estes podem ter evoluído de um ancestral em comum. De acordo com o critério molecular para demarcação de espécies de Potyvirus, 82 % e 76 % de identidade na comparação da seqüência de aminoácidos e nucleotídeos da CP, respectivamente, o isolado C17 de mandioquinha-salsa pode ser considerado como uma nova espécie de vírus pertencente ao gênero Potyvirus, e o nome Arracacha mottle virus é proposto. Com o intuito de desenvolver uma ferramenta de detecção mais sensível que a sorologia, primers específicos foram desenhados e avaliados. Um par de primers mostrou excelentes resultados de especificidade e sensibilidade, sendo recomendados para o uso em testes de detecção em situações de necessidade de alta sensibilidade. A seqüência da região 3’ terminal genômica deste vírus foi bastante distinta dos demais potyvírus, portanto a clonagem do genoma para sequenciamento completo foi realizada. Utilizou-se a estratégia de RT-PCR com primers desenhados em regiões conservadas do genoma dos potyvírus mais próximos. Um total de dez clones foram obtidos, o que corresponde a 90% do genoma do vírus. A seqüência nucleotídica destes clones estão sendo determinados por primer walking. Até o momento, 70 % da seqüência de nucleotídeos do genoma já foi determinada. Este é o primeiro relato de identificação e caracterização de vírus infectando mandioquinha-salsa no Brasil. ________________________________________________________________________________ ABSTRACT / Arracacha plant is vegetatively propagated and during its continuous cultivation accumulation of systemic pathogens, such as viruses, can occur resulting in decreased production. In Brazil, it is common to observe arracacha plants with symptoms of mosaic, mottling and leaf deformation, similar to virus infection. Evidences of virus occurrence have been reported, but their identification and characterization were not carried out. The objective of this study was to characterize at the biological, serological and molecular level a virus isolated from arracacha, and to develop detection techniques. The virus isolate was obtained from an arracacha plant collected for micropropagation studies. This isolate was named C17, and selected for characterization. This virus caused in Nicotiana benthamiana plants, the maintenance host, symptoms of leaf deformation, blistering and mosaic. Biological isolation was conducted from the extract of the original arracacha plant inoculated in Chenopodium quinoa leaves. This isolate had a narrow host range and was transmitted by aphids in a non-persistent manner. Viral particles were purified and EM analysis revealed flexuous and filamentous particles, typical of Potyvirus species. A SDSPAGE revealed a coat protein of ca. 33 kDa, which was specifically recognized by the C17 polyclonal antiserum by Western blotting. This antiserum was produced in rabbits from purified particles, and was shown to be highly specificic and sensitive. Samples of the germplasm bank of arracacha of Embrapa Hortaliças were tested by ELISA. A total of 93% were positive for the presence of C17, demonstrating the applicability of this antiserum in diagnostic tests in field collected samples. The 3’ genomic portion of the virus genome (1,7 kb) was cloned by RT-PCR and sequenced in an automated sequencer. Nucleotide sequence analysis of the cloned fragment showed a partial ORF (without the start codon) encoding the 3’ portion of the NIb protein (viral polymerase), and the coat protein (CP), followed by a 3' untranslated region (3'UTR). The CP and 3’ UTR nucleotide sequence shared low identity with other potyvirus sequences in public databases. The highest nucleotide identity was 63 % with Araujia mosaic virus (ArjMV), while the highest amino acid identity (60 %) with Narcissus late season yellows virus (NLSYV). The phylogenetic analysis confirmed the clustering of C17 isolate with NLSYV and suggested that they may have evolved from a common ancestral. According to the species demarcation criterion for Potyvirus species (82% and 76% identity of CP amino acid and nucleotide sequence, respectively), C17 isolate can be considered as a novel virus belonging to the Potyvirus genus. The name Arracacha mottle virus is proposed. In order to develop a detection tool more sensitive than serology, specific primers were designed and evaluated for the use in RT-PCR. One primer pair showed excellent specificity and sensitivity results and is recommended for application in detection tests when high sensitivity is needed. The sequence of the 3' terminal end of this virus was quite different from other potyviruses, hence complete genome cloning was attempted. The strategy used was based on RT-PCR with primers designed towards conserved regions of the potyvirus genome. A total of ten partial clones were obtained, which corresponded to 90% of the viral genome. Inserts of these clones are being sequenced by primer walking. To date, 70% of the nucleotide sequence has already been determined. This is the first report of the identification and characterization of a virus infecting arracacha in Brazil.

The possible existence of variability in the reaction of PMV in soybean, which could lead to resistance breaking strains, was investigated in this study. The specific research objectives were to provide evidence of PMV strains in soybean, and to characterize the PMV strains using aphid transmission efficiency and serological relationships. Twelve PMV isolates were placed in five strain groups (P1-P5) based on symptom expression in the soybean cultivars Lee 68, York, and Virginia. Aphid transmission efficiency of the PMV strains by the green peach aphid (Myzus persicae Sulz.) was determined by counting infected test plants following natural feeding by aphids exposed to virus infected source plants. Transmission efficiency from pea (Pisum sativum L.) source plants to pea test plants was 11 to 36% and varied with strains. Pea to soybean and soybean to soybean transmission was 5 to 29% and 20 to 58%, respectively. From these results, the 5 strains could be classified in 2 groups. Two techniques, SDS-immunodiffusion (sodium dodecyl sulfate) and ELISA (enzyme-linked immunosorbent assay), were used to investigate the serological relatedness of the PMV strains. Results indicated that all the strains were serologically closely related. / Ph. D.

Made available in DSpace on 2015-03-26T13:37:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1056837 bytes, checksum: 147af3aea5c0fd4d6961921a210a152c (MD5) Previous issue date: 2012-02-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to productivity losses in the main producing regions of Brazil. The disease was first recorded in the Midwest region of Brazil in the 2000/01 soybean season and rapidly spread throughout the country in the following years. CpMMV is usually hard to diagnose due to its wide range of symptoms while the occurrence of asymptomatic soybean cultivars complicates genotypic selection in breeding programs. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein sequence of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a 6 His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/Tof mass spectrometer analyses. New Zealand rabbits were immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antisera was shown by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection. / O Cowpea mild mottle virus (CpMMV), agente causal da necrose da haste, tem chamado a atenção dos produtores de soja nos últimos anos devido às perdas de produtividade geradas nas principais regiões produtoras do Brasil. A doença foi relatada pela primeira vez na região Centro-Oeste do Brasil, na safra de 2000/01, e rapidamente se espalhou por todo o país nos anos seguintes. Além disso, tem sido relatada a ocorrência de cultivares de soja assintomáticas, o que dificulta a seleção de genótipos para programas de melhoramento. Os métodos sorológicos para a detecção viral requerem o uso de um antissoro de boa qualidade para alcançar especificidade e sensibilidade. A sequência completa da capa proteica de um isolado brasileiro de CpMMV (Bahia) foi clonado em um vetor de expressão bacteriano e transformado em Escherichia coli BL21: DE3 para expressão in vitro. A proteína capsidial ligada a uma cauda de histidina foi purificada sob condições desnaturantes através de cromatografia de afinidade, utilizando uma resina Ni-NTA. Depois da renaturação, a integridade e a identidade da proteína recombinante purificada foi confirmada por SDS-PAGE e análise de espectrometria de massa (MALDI-ToF/ToF). Dois coelhos da raça Nova Zelândia foram imunizados com quantidades crescentes da proteína recombinante. A especificidade e sensibilidade do antissoro foram avaliadas por Western blot e ELISA indireto. O antissoro policlonal a partir da proteína recombinante da capa proteica provou ser uma ferramenta confiável e eficiente para a detecção CpMMV.

The aim of this work was to develop a solid knowledge on formulation effects controlling offset ink-paper coating adhesion and to identify key factors of the coating and printing process affecting it. Focus lay on comprehending the impact of pigment dispersant on ink-paper coating adhesion and ultimately on the print quality of offset prints. The work covers laboratory studies, a pilot coating trial designed to produce coated material with a span in surface chemistry and structure, and an industrial offset printing trial. The lab scale studies quantified ink-paper coating adhesion failure during ink setting with a developed laboratory procedure based on the Ink-Surface Interaction Tester (ISIT) and image analysis. Additional polyacrylate dispersant resulted in slower ink setting and reduced ink-paper coating adhesion, with a dependence on its state of salt neutralisation and cation exchange, mainly in the presence of moisture/liquid water. The industrial printing trial on pilot coated papers was designed to study how these laboratory findings affected full scale offset print quality. These trials confirmed the dispersant-sensitive effect on ink-paper coating adhesion, especially at high water feeds. Evaluation of prints from the printing trial resulted in two fundamentally different types of ink adhesion failure being identified. The first type being traditional ink refusal, and the second type being a novel mechanism referred to as ink-lift-off adhesion failure. Ink-lift-off adhesion failure occurs when ink is initially deposited on the paper but then lifted off in a subsequent print unit. In this work, ink adhesion failure by this ink-lift-off mechanism was observed to occur more often than failure due to ink refusal. Print quality evaluation of the industrial prints suggested that water induced mottle was caused by a combination of ink-surface adhesion failure, creating white spots on the print, together with variation in ink layer thickness due to emulsified ink. / <p>QC 20161019</p>

Le virus de la panachure jaune du riz (virus RYMV pour Rice Yellow Mottle Virus) infecte principalement le genre Oryza et provoque d'importants dégâts sur les cultures de riz en Afrique. Bien que son génome soit rudimentaire, ce virus code des protéines essentielles pour son maintien chez l’hôte en dépit des mécanismes de défense de la plante. Les travaux récents de l’équipe ont permis d’identifier la protéine P1 codée par ce virus comme une protéine qui pourrait, grâce à sa propriété de suppresseur de RNA silencing, permettre au virus de contourner un mécanisme de défense essentiel de l’hôte et permettre au virus de perpétuer son cycle viral. Peu de données concernant les mécanismes d’action de la protéine P1 sont disponibles à ce jour. Le travail entrepris au cours de ma thèse a donc consisté à compléter les connaissances sur la biochimie de cette protéine, à définir sa structure tridimensionnelle et à mettre à jour sa localisation sub cellulaire afin de révéler des propriétés qui pourraient nous permettre non seulement de mieux comprendre comment cette protéine opère ses fonctions mais également de définir des méthodes de lutte adéquates contre ce virus. Ainsi, je montre que la protéine P1 constitue une nouvelle famille de protéine à doigt de zinc possédant une structure 3D inédite composée d’un premier domaine impliqué dans la dimérisation de la protéine et dans des interactions avec des ligands dont certains pourraient provenir de la plante hôte. Mon travail permet également d’identifier un deuxième domaine senseur de l’état redox au sein de la protéine qui lui permet probablement de sonder l’état de la plante pendant l’infection virale et d’adapter ses conformations pour assurer ses fonctions. Finalement, une approche par mutagénèse sur la protéine P1 assistée par la nouvelle structure 3D démontre qu’il est désormais possible d’identifier les résidus essentiels à la protéine pour sa participation dans l’infection virale. Ce travail ouvre donc de nombreuses perspectives pour de futures études de mécanistique sur ces domaines-clé de la protéine, ainsi que pour des études sur sa diversité génétique au sein des très nombreux isolats du virus RYMV en Afrique. / The virus of rice yellow mottle virus (RYMV for Rice Yellow Mottle Virus) mainly infects the genus Oryza and causes significant damage to rice crops in Africa. Although its genome is rudimentary, this virus code essential proteins for its maintenance in the host despite the defense mechanisms of the plant. Recent work by the team has identified the P1 protein encoded by the virus as a protein that could, through its ownership of RNA silencing suppressor, allow the virus to bypass an essential defense mechanism of the host and allow the virus to perpetuate its viral cycle. Little data on the mechanisms of action of the P1 protein is available to date. The work undertaken during my thesis was therefore to supplement the knowledge of the biochemistry of this protein, to define its three-dimensional structure and update its sub cellular localization to reveal properties that could enable us not only to understand how this protein works its functions but also to define methods of adequate response against the virus. Thus, I show that the P1 protein is a new zinc finger protein family having a unique 3D structure consisting of a first domain involved in the dimerization of the protein and in interactions with ligands some of which may originate from the plant host. My work also identifies a second sensor field in the redox state of the protein that probably allows him to probe the state of the plant during viral infection and adapt its conformation to conduct their duties. Finally, a mutagenesis approach to P1 assisted by the new 3D protein structure shows that it is now possible to identify critical residues in the protein for its participation in the viral infection. This work thus opens up many possibilities for future mechanistic studies on these key areas of the protein, as well as for studies of genetic diversity within many RYMV isolates of virus in Africa

X-ray crystallography is one of the most powerful tools for the elucidation of the structure of biological macromolecules such as proteins and viruses. Crystallographic techniques are extensively used for investigations on protein structure, ligand-binding, mechanisms of enzyme catalyzed reactions, protein-protein interactions, role of metal ions in protein structure and function, structure of multi-enzyme complexes and viruses, protein dynamics and for a myriad other problems in structural biology. Crystallographic studies are essential for understanding the intricate details of the mechanism of action of enzymes at molecular level. Understanding the subtle differences between the pathogenic enzymes and host enzymes is necessary for the design of inhibitor molecules that specifically inhibit parasite enzymes. The current thesis deals with the application of biochemical and crystallographic techniques for understanding the structure and function of proteins from two pathogenic organisms – a plant virus Physalis Mottle Virus (PhMV), and a pathogenic bacterium, Salmonella typhimurium and also stress induced proteins from Oryza sativa. The thesis has been divided into seven chapters, with the first four chapters describing the work carried out on PhMV, while the rest of the chapters deal with the studies on stress response proteins from Oryza sativa and Salmonella typhimurium. The first part of the thesis deals with studies on viral capsids. Viruses are obligate parasites that have proteinaceous capsids enclosing the genetic material, which, in the case of small plant viruses, is invariably ss-RNA. X-ray diffraction studies on single crystals of viruses enable visualization of the structures of intact virus particles at near-atomic resolution. These studies provide detailed information regarding the coat protein folding, molecular interactions between protein subunits, flexibility of the N-and C-terminal segments and their probable importance in viral assembly, role of RNA in capsid assembly, nucleic acid (RNA)-protein interactions, the capsid structure and mechanism of assembly and disassembly. The present thesis deals with the capsid structure and analysis of the coat protein (CP) recombinant mutants of PhMV. Virus assembly, one of the important steps in the life cycle of a virus, involves specific interactions between the structural protein and cognate viral genome. This is a complex process that requires precise protein-protein and protein nucleic acid interactions. In fact, most of the biological functional units such as ribosomes and proteosomes also require highly co-ordinated macromolecular interactions for their functional expression. Viruses being simple in their architecture, serve as excellent model systems to understand mechanism of macromolecular assembly and provide necessary information for the development of antiviral therapeutics, especially in animal viruses. PhMV is a plant virus infecting several members of Solanaceae family. It belongs to the tymoviridae group of single stranded RNA viruses. Its genome is encapsidated in a shell comprising of 180 (architecture based on T = 3 icosahedral lattice) chemically identical coat protein (CP) subunits (~ 20,000Da) arranged with icosahedral symmetry. In an earlier phase of work, PhMV purified from infected plant leaves was crystallized in the space group R3 (a = 294.56 Å,  = 59.86). X-ray diffraction data to 3.8 Å resolution were recorded on films by screenless oscillation photography. Using this data of severely limited quality and poor completion (40%), the structure PhMV was determined by molecular replacement using the related turnip yellow mosaic virus (TYMV) structure as the phasing model. There was therefore a need to re-determine and improve the structure, which could be useful for understanding the earlier detailed studies on its biophysical properties. As a continuation of these studies, the present investigations were conceived with the goal of determining the natural top and bottom component capsid structures of PhMV. Investigations were also carried out to examine the possibility of enhancing the diffraction quality of PhMV crystals. The thesis begins with a review of the current literature on the available crystal structures of viruses and their implications for capsid assembly (chapter I). All experimental and computational methods used during the course of investigations are described in chapter II, as most of these are applicable to all the structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and analysis. Chapter III describes structural studies on top and bottom components of PhMV. Purified tymoviruses including PhMV are found to contain two classes of particles that sediment at different velocities through sucrose gradients and are called the top (sedimentation coefficient 54 Svedberg units(S)) and the bottom (115S) components. The top component particles are either devoid of RNA or contain only a small subgenomic RNA (5%) while the bottom component particles contain the full length genomic RNA. Only the bottom component is infectious. The top and bottom components were separately crystallized in P1 and R3 space groups, respectively. It is of interest to note that crystals of the bottom component obtained earlier belonged to R3 space group while recombinant capsids that lack of full length RNA as in natural top component crystallized in the P1 space group. A polyalanine model of the homologous TYMV was used as the phasing model to determine the structures of these particles by molecular replacement using the program AMoRe. The refinement of top and bottom component capsid structures were carried out using CNS version 1.1 and the polypeptide models were built into the final electron-density map using the interactive graphics program O. The quality of the map was sufficient for building the model and unambiguous positioning of the side chains. There is a significant difference in the radius of the top and bottom component capsids, the top component being 5 Å larger in radius. Thus, RNA makes the capsid more compact, even though RNA is not a pre-requisite for capsid assembly. Partially ordered RNA was observed in the bottom component. The refined models could form the basis for understanding the architecture, protein-protein interactions, protein-nucleic acid interactions, stability and assembly of PhMV. Chapter IV provides a detailed description of the mutations carried out on PhMV coat protein towards enhancing the diffraction quality of crystals. The gene coding for PhMV coat protein (PhMVCP) and several of its deletion and substitution mutants were originally cloned in pRSETC and pET-21 vectors by Mira Sastri and Uma Shankar in Prof. Savithri’s laboratory at the Department of Biochemistry. It was observed that the recombinant intact coat protein and several mutants lacking up to 30 amino acids from the N-terminal end could assemble into empty shells resembling the natural top component. None of these deletion mutants crystallized in forms that diffracted to high resolution. Based on the intersubunit contacts observed, three more site-specific mutants were designed. These three mutants were expressed in BL21 (DE3), purified and crystallized. Even these mutant crystals did not diffract to high resolution. The polypeptide fold of PhMV coat protein therefore was carefully examined for probable reasons. It was found that PhMV subunit has three major cavities. Three cavities are likely to increase the flexibility of protein subunits, which in turn may result in crystals of poor quality. Mutations V52W, S158Q and A160L were shown to fill up these cavities and with the view of obtaining better crystals. These site specific mutations were carried out the mutant proteins were purified. It was shown that the recombinant capsids are stable and possess T=3 architecture. Two mutants were crystallized and a data set for V52W extending to 6.0 Å resolution could be collected. Due to the limited resolution, further work was not pursued. It is plausible that the triple mutant will diffract to higher resolution. The second part of the thesis deals with stress response proteins from Oryza sativa and Salmonella typhimurium. It is known that viral infection and abiotic and biotic stresses induce a network of proteins in plants. Chapter V presents a review of the current literature on stress proteins, focusing mainly on Oryza sativa and S. typhimurium stress response proteins. Chapter VI describes the over expression of stress proteins SAP1 and SAP2 from rice. These stress related proteins confer tolerance to cold, dehydration and salt stress in rice. These proteins have been cloned in the expression vector pEt-28(a) and expressed in E. coli strain BL21 CodonPlus(DE3)RIL. The proteins were purified and crystallization trials were made. However, there were no hits. In an attempt to get crystals, nine deletion constructs of SAP1 were designed eliminating potentially disordered and unfolded regions based on a bioinformatics analysis. Crystallization trails are being carried out on three of the constructs. Structural studies on a universal stress protein from Salmonella typhimurium, which shares homology with the rice universal stress proteins, was initiated. Apart from this, several other stress related proteins of Salmonella typhimurium have also been selected for structural and functional studies. These include YdaA, YbdQ, Yic, Ynaf, Yec, Spy and Usb. All these were cloned and expressed in E. coli. Out of seven proteins, Ynaf, YdaA and YbdQ were found in the soluble fraction and were expressed in quantities suitable for structural studies. I could crystallize YdaA and Ynaf. X-ray diffraction data to resolutions of 3.6 Å and 2.3 Å were collected on crystals of YdaA and YnaF, respectively. A tentative structure of YnaF has been obtained. Further attempts to determine these structures are in progress. Biophysical, Biochemical functional characterization of YdaA and YnaF proteins are described. Structural studies on mannose-6-phosphate isomerase, an enzyme related to stress regulatory proteins from S. typhimurium are dealt with in Chapter VII. Mannose 6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The structure could be solved in its apo and holo forms (with two different metal atoms, Y3+ and Zn2+), and complexed with the cyclic form of the substrate fructose 6-phosphate (F6P) and Zn2+. Isomerization involves acid/base catalysis with proton transfer between C1 and C2 atoms of the substrate. Lys 132, His 131, His 99 and Asp 270 are close to the substrate and are likely to be the residues involved in proton transfer. Interactions observed at the active site suggest that the ring opening step is catalyzed by His 99 and Asp 270. An active site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. The metal ion is not close to the substrate atoms involved in proton transfer. Binding of the metal induces structural order in the loop consisting of residues 50-54. Hence, the metal atom does not appear to play a direct role in catalysis, but is probably important for maintaining the architecture of the active site. Based on these structures and earlier biochemical work, a probable isomerization mechanism has been proposed. The thesis concludes with a brief discussion on the future prospects of the work. The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis:

Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. Soybean yield losses of 10-40% have been reported as a consequence of BPMV infection. The complete nucleotide sequences of two strains, K-Ha1 and K-Ho1, were determined. Field isolates of BPMV were classified into two distinct subgroups (I and II) based on slot blot hybridization and sequence analyses. Full-length cDNA clones from which infectious transcripts can be produced were constructed for strains K-G7, K-Ho1 and K-Ha1. Whereas strains K-Ha1 and K-G7 induced mild or moderate symptoms in infected soybean plants, strain K-Ho1 produced very severe symptoms. Symptom severity was mapped to RNA1. Chimeric RNA1 constructs were generated by exchanging full or partial coding regions of the five RNA1-encoded mature proteins between the full-length cDNA clones of the three RNA1s and the resultant transcripts were inoculated onto soybean. The results showed that the coding regions of the protease co-factor (Co-pro) and the putative helicase (Hel) are determinants of symptom severity. Although symptom severity correlated well with accumulation of viral RNA, neither the Co-pro nor Hel protein could be demonstrated as a suppressor of RNA silencing. Furthermore, separate expression of the Co-pro or Hel proteins from a PVX vector induced necrosis on the inoculated leaves of Nicotiana benthamiana. Characterization of BPMV K-Ho1 indicated that it is a diploid reassortant, containing two distinct types of RNA1s and one type of RNA2. Examination of field isolates from various locations in the United States and Canada revealed that diploid reassortants are of frequent occurrence in natural populations of BPMV. The vary severe symptoms induced by BPMV K-Ho1 can be mimicked by inoculation of plants with a mixture of RNA1 transcripts from two distinct strain subgroups and RNA2 transcript from either subgroup. Plants inoculated with a mixture of transcripts containing two types of RNA1 from the same strain subgroup did not produce very severe symptoms. These are due to interactions between two distinct types of RNA1s. At present, no soybean cultivars with resistance to BPMV are commercially available. Therefore, the feasibility of cross protection as an alternative disease management strategy was studied. Two mild strains of BPMV (K-Da1 and K-Ha1), belonging to subgroup II, were tested for their ability to protect infected plants against a severe strain (K-Ho1). Inoculation of the soybean cultivar Essex on the primary leaves with either of the two mild strains conferred complete protection against challenge inoculation with the severe strain K-Ho1, regardless of the timing of challenge inoculation. Cross-protection was evident regardless of whether virions or BPMV-RNA were used as inocula. Cross protection was independent of the soybean cultivar used and method of virus inoculation, sap-inoculation or by the bean leaf beetle, vector of BPMV. Protection was complete and durable.

<p>What is meant with print quality, what makes people perceive the quality of an image in a certain way? An inquiry was made about what the parameters are that strongly affect the perception of digital printed images. </p><p>A subjective test and some measurements make the basis for the thesis. The goal was to find a tool to predict perceived image quality when investigating the connections between the subjective test and the measurements. </p><p>Some suitable images were chosen, with a variety of motifs. A test panel consisting of people that are used to observe image quality answered questions about the perception of the quality. Measurements were made on a special test form to get information about the six different printers used in the investigation. </p><p>One of the discoveries was made when two images with the same colorful motif were compared. The first image got a much higher grade for general quality than the second image, even though the second image was printed with a printer that had a larger color gamut. The reason of this is that the first image consists of more saturated colors, and the second image has more details. The human eye perceives the more saturated image to be better than the image with more details. Another discovery was the correlation between the perceived general quality of a colored image and the perceived color gamut. One conclusion was that a great difference between two calculated color gamuts resulted in a large difference in perception of the color gamuts. A discovery of an image with very few colors and many glossy surfaces was that print mottle and sharpness are strictly connected to the general quality.</p>

The objective of this thesis is to better understand the impact of paper and printer types on xerographic print quality. To achieve this objective, commercially printed samples comprising of ten different paper substrates printed using three different xerographic printers were examined. The print quality of these samples was assessed in terms of print microgloss and its nonuniformity, print density, print and gloss mottle, print roughness, and visual ranking. This study showed that print mottle conducted by Fast Fourier Transform produced the best correlation with visual ranking at the size range of 0.1 - 1mm, while print gloss mottle was found to affect print quality regardless of the mottle size. Brightness, opacity, basis weight, gloss 75, and roughness of these paper substrates were found to have the most significant effect on print quality. All of the optical properties of paper included in this analysis showed a strong correlation to print quality.