Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Hierarchical information transfer is investigated as a tool to prepare well‐defined nanostructures with high aspect ratios, via the self‐assembly of helically folding poly(paraaryltriazole) (P(p‐AT)) foldamers. A novel ‘helicity codon’ based on the 1,4‐linkage geometry in 1,4‐aryl‐disubstituted‐1,2,3‐ triazoles is developed. Helical folding is induced exclusively by directing all triazole moieties into a cisoid configuration. By linking the triazole rings in a para fashion about the aryl moiety, this helicity codon codes for a helix with a large internal cavity of ~ 3 nm. One turn of the putative helical conformation requires 14 repeat units and the helical pitch is ~ 0.38 nm. The aryltriazole backbone is appended with amphiphilic oligo(ethylene glycol) (oEG) units which have the dual roles of imparting solubility as well as instigating a solvophobic helical folding in solvents which poorly solvate the hydrophobic arytriazole backbone but, solvate the side chains fully. The helix interior is hydrophobic and the exterior is amphiphilic. A true polymer synthesis approach to the foldamer synthesis, based on the copper catalysed azide‐alkyne cycloaddition (CuAAC) AB step growth polymerization system, is developed. This is preceded by a facile synthetic protocol for the AB monomers. The subsequent P(p‐ AT)s have high molecular weights ensuring several turns in the helical foldamer. A DMF/H2O good solvent/bad solvent system is established. Twist sense bias in the helical foldamers is successfully imparted by installing enantiopure chiral oEG side chains. Spectroscopic signatures for the solvent dependent coil to helix transition are established enabling the tracking of the conformational transitions from primary to secondary and finally tertiary structure. Conclusive evidence for the formation of stable, long stacked helical columns, in the solution state, is provided via cryo‐TEM. The helical stacks are several microns long, but of random lengths and do not intertwine but rather run parallel to each other. The helical stacks, however, have indeterminate lengths. Control over the length and chirality of the self‐assembled helical stacks is successfully imparted by using a template which mimics the role of ribonucleic acid (RNA) in tobacco mosaic virus (TMV). The template used is the hydrophobic α‐helical polypeptide poly(γ‐ benzyl‐L‐glutamate) (PBLG). Self‐assembly is driven by solvophobicity in a DMF/H2O system, the PBLG template being encapsulated inside the hydrophobic cavities of the stacked/selfassembled helical foldamers. Information from the template, i.e. length and chirality, is used to control the length and the chirality of the stacked/self‐assembled construct. The templated self‐assembly process is solvent dependent. When carried out in the solvent regime at the coil to helix transition mid‐point of the foldamer host, system operates under a dynamic equilibrium. Under these conditions, the self‐assembly process is shown to take place between two distinct states, the foldamer helices and the helical template, the template threading through the foldamer helices. The resulting self‐assembled construct has a pseudo‐rotaxane architecture. Under dynamic equilibrium conditions, temperature induced dis‐assembly of the templated assembled construct, is shown to be a cooperative process, whilst re‐assembly is characterized by a large hysteresis. By increasing the volume fraction of water, the solvophobic character of the system is increased and template assembled construct is better stabilised. The assembly system, however, loses its dynamic equilibrium character and falls into kinetic traps. Temperature induced de‐threading, of the foldamer helices, becomes less favourable and loses its cooperative character although the hysteresis loop is reduced. / AFRIKAANSE OPSOMMING: Hiërargiese inligtingsoordrag is bestudeer as ‘n hulpmiddel om goed gedefinieerde nanostrukture met ‘n goeie beeldverhouding voor te berei. Die nanostrukture word voorberei deur middel van self‐samestelling van heliese vouing van poli(para‐arieltriasool) (P(p‐AT)) ‘foldamers’. ‘n Nuwe heliese‐kodon gebaseer op die 1,4 koppelingsgeometrie in 1,4 arieldigesubstitueerde‐ 1,2,3‐triasool is ontwikkel. Heliese vouing word uitsluitlik geïnduseer as al die triasole in die sis konfigurasie is. Deur die triasole in ‘n para konfigurasie te bind, kodeer die heliese kodon vir ‘n heliks met ‘n groot interne kanaal van ~ 3 nm. Een draai van die heliks benodig 14 herhalende eenhede en die heliese gradiënt ~ 0.38 nm. Amfifiliese oligo(etileen glikol) (oEG) eenhede is aan die arieltriasoolruggraat aangeheg. Hierdie aanhegting van oEG eenhede bevorder oplosbaarheid en dit induseer ‘n solvofobiese heliese vouing in oplosmiddels wat nie die hidrofobiese arieltriasoolruggraat oplos nie, maar wel die sy‐kettings volledig oplos. Die binnekant van die heliks is hidrofobies en die buitekant is amfifilies. ‘n Polimeersintese benadering tot die ‘foldamer’ sintese (gebaseer op die koper gekataliseerde siklo‐addisie reaksie tussen ‘n asied en ‘n alkyn) AB stapsgewyse groei polimerisasiestelsel, is ontwikkel. Dit is voorafgegaan deur ‘n geskikte sintetiese protokol vir die AB monomere. Die daaropvolgende P(p‐AT) het ‘n hoë molekulêre massa wat verseker dat daar ‘n hele paar draaie in die heliese ‘foldamer’ is. ‘n DMF/H2O goeie oplosmiddel/ swak oplosmiddel sisteem is vasgestel. Draaiing van die heliks na ‘n spesifieke kant alleenlik is suksesvol geïnduseer deur die toevoeging van suiwer enantiomere van die chirale oEG sykettings. Spektroskopiese handtekeninge van die oplosmiddel‐afhanklike ketting tot heliks transformasie word vasgestel sodat die oorgangstoestande gevolg kan word vanaf primêre tot sekondêre en uiteindelik tesiêre struktuur. Beslissende bewyse vir die formasie van stabiele, lang gestapelde heliese kolomme in die opgeloste toestand is bewys met cryo‐TEM. Die heliese stapels is verskeie mikron lank, maar het verskillende lengtes. Die heliese stapels is parallel aan mekaar en oorvleuel nie. Die lengte van die heliese stapels is egter onbepaalbaar. Beheer oor die lengte en chiraliteit van die self‐samestellende heliese stapels is verkry deur gebruik te maak van ‘n templaat wat die rol van ribonukleïensuur (RNS) in die tabakmosaïekvirus (TMV) naboots. Hidrofobiese α‐heliese polipeptied poli(γ‐bensiel‐Lglutamaat) (PBLG) is gebruik as die templaat. Self‐samestelling word gedryf deur solvofobisiteit in ‘n DMF/H2O stelsel, met die PBLG templaat wat dan geënkapsuleer word binne die hidrofobiese holtes van die gestapelde/ self‐saamgestelde heliese ‘foldamers’. Die lengte en die chiraliteit van die templaat word gebruik om die lengte en chiraliteit van die gestapelde helikse te beheer. Die templaatbemiddelde self‐samestellende proses is afhanklik van die oplosmiddel. Die stelsel is by ‘n dinamiese ewewig wanneer, uitgevoer in ‘n oplosmiddel, die ketting na heliks oorgang die middelpunt van die ‘foldamer’ gasheer bereik het. By hierdie omstandighede vind die self‐samestellende proses plaas tussen twee afsonderlike toestande nl. die ‘foldamer’ helikse en die heliese templaat, en die templaat wat vleg deur die ‘foldamer’ helikse vleg. Die gevolglike struktuur het ‘n pseudo‐rotaxane argitektuur. By dinamiese ewewigstoestande veroorsaak temperatuur dat die self‐samestellende templaatstrukture weer disintegreer. Hierdie is ‘n koöperatiewe proses terwyl die hersamestelling gekarakteriseer word deur ‘n sloerende proses. Deur die waterfraksie te vermeerder, word die solvofobiese karakter van die sisteem verhoog en die templaat selfsamestellende struktuur beter gestabiliseer. Die samestellingsproses verloor egter sy dinamiese ewewigkarakter en val in kinetiese slaggate. Temperatuur geïnduseerde disintegrasie van die foldamer helikse word minder gunstig en dit verloor die koöperatiewe karakter alhoewel die sloering verminder is.

This thesis addresses the design and use of suitable nanomaterials and surface acoustic wave sensors for hydrogen detection and sensing. Nanotechnology is aimed at design and synthesis of novel nanoscale materials. These materials could find uses in the design of optical, biomedical and electronic devices. One such example of a nanoscale biological system is a virus. Viruses have been given a lot of attention for assembly of nanoelectronic materials. The tobacco mosaic virus (TMV) used in this research represents an inexpensive and renewable biotemplate that can be easily functionalized for the synthesis of nanomaterials. Strains of this virus have been previously coated with metals, silica or semiconductor materials with potential applications in the assembly of nanostructures and nanoelectronic circuits. Carbon nanotubes are another set of well-characterized nanoscale materials which have been widely investigated to put their physical and chemical properties to use in design of transistors, gas sensors, hydrogen storage cells, etc. Palladium is a well-known material for detection of hydrogen. The processes of absorption and desorption are known to be reversible and are known to produce changes in density, elastic properties and conductivity of the film. Despite these advantages, palladium films are known to suffer from problems of peeling and cracking in hydrogen sensor applications. They are also required to be cycled for a few times with hydrogen before they give reproducible responses. The work presented in this thesis, takes concepts from previous hydrogen sensing techniques and applies them to two nanoengineered particles (Pd coated TMV and Pd coated SWNTs) as SAW resonator sensing materials. Possible sensing enhancements to be gained by using these nanomaterial sensing layers are investigated. SAW resonators were coated with these two different nano-structured sensing layers (Pd-TMV and Pd-SWNT) which produced differently useful hydrogen sensor responses. The Pd-TMV coated resonator responded to hydrogen with nearly constant increases in frequency as compared to the Pd-SWNT coated device, which responded with concentration-dependent decreases in frequency of greater magnitude upon hydrogen exposure. The former behavior is more associated with acousto-electric phenomena in SAW devices and the later with mass loading. The 99% response times were 30-40 seconds for the Pd-TMV sensing layer and approximately 150 seconds for the Pd-SWNT layer. Both the films showed high robustness and reversibility at room temperature. When the Pd film was exposed to hydrogen it was observed that it produced decreases in frequency to hydrogen challenges, conforming to mass loading effect. It was also observed that the Pd film started degrading with repeated exposure to hydrogen, with shifts after each exposure going smaller and smaller.

Thesis (Ph.D.)--Baylor University, 2005. / Includes bibliographical references (p. 145-157).

Verticillium is a soil-borne fungus that is one of the world's foremost plant pathogens. Commercial plant grafting suggests that resistance occurs in the root; this conflicts with decades of research indicating that resistance occurs in the stem. The goal of this thesis work was to use an alternative approach to determine the location of resistance by expressing the Ve1 gene using organ-specific promoters. Promoter sequences for the stem-specific gene, Ribulose 1,5-bisphosphate carboxylase oxygenase small chain 2A (Rbsc2A), and root-specific gene, Tobacco Mosaic Virus Induced (TMVi) were taken from the Sol Genomics Network (SGN) database, cloned into constructs with the Ve1 gene and susceptible tomato germplasm was transformed using Agrobacterium tumefaciens. Preliminary results suggest that resistance may not be localized and expression of the Ve1 gene in either the root or the stem is sufficient to develop whole plant resistance to the Verticillium pathogen.

The Wheeler Orchid Collection is home to some of the most endangered species of orchids in the world. This fantastic reservoir of endangered species has been enhanced and broadened by its function as a plant rescue station for the U.S. customs service. Unfortunately, this responsibility increases the risk of bringing orchids, which harbor contageous diseases, into the greenhouse where sap transmitted diseases such as the Tobacco Mosaic Virus (TMV), can run rampant. Although manipulation of orchid characteristics is typically done by classical plant breeding techniques, genetic engineering is emerging as a useful technique for the introduction of desirable traits into the orchid genome. Through the use of genetic engineering techniques it may be possible to mitigate the symptoms associated with this destructive virus. Virus resistance may be achieved through the expression of either the sense or antisense viral coat protein gene in orchid tissues if an efficient means of orchid transformation is developed. In this research two transformation protocols were examined for their ability to efficiently transform orchid tissue. The first transformation protocol explored utilized the native ability of Aq bacterium tumefaciens to incorporate DNA into host plants to achieve transformation. The second mechanism explored was particle bombardment transformation.Many strains of A. tumefaciens were employed using direct exposure of Cattleya_ orchid protocorm and callus tissue. Particle bombardment using DNA coated 0.5 um diameter tungsten particles and high pressure helium tank acceleration was employed. The particle bombardment procedure employed the pG35barB plasmid which confers herbicide resistance to the herbicide basta when integrated and expressed in plant tissues.GUS fluorescence assays and PCR analysis indicate that T-DNA is present in orchid tissues, while Southern blot analysis was unable to display that integration had occurred. Particle bombardment yielded herbicide resistant orchid tissues which have yet to be analyzed by Southern blot analysis to confirm integration due to limited tissue quantities. / Department of Biology

A major goal of our laboratory is to confer resistence specifically to the Odontoglossum ringspot virus [ORSV; sometimes referred to as tobacco mosaic virus strain O (TMV-O)] in orchids. The chosen strategy may also provide cross-protection to other pathogens. The experimental design for the entire project is presented here along with the results obtained in several preliminary experiments performed in this research. Our approach involved RT-PCR amplification of the viral coat protein gene with gene-specific primers and digestion of the cDNAs into oligonucleotides. These fragments were cloned into the selectable vector pG35barB (which confers herbicide resistence) in both sense and antisense orientations. The cloned DNA was coated with tungsten beads and shot into orchid callus tissue using a makeshift biolistic gun. Tranformant callus cells were selected for by herbicide resistance. Unfortunately the potential transformants became contaminated with fungus and could nto be analyzed to determine which oligonucleotide was received and the effect each oligonucleotide had on pathogen resistance. Due to the uncertainty of the relatedness between ORSV and TMV-O, we also sequenced the coat protein gene of TMV-O and compared the amino acid sequence with those of several strains of ORSV: the Japanese strain had the highest percent amino acid similarity (99.4%), the Type strain the second highest (98.7%), and the Korean strain the lowest (96.9%). It was concluded that TMV-O is most likely one strain of ORSV, the Japanese strain. / Department of Biology

Thesis (M.S.)--University of Wisconsin--Madison, 1982. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 53-56).

Orientador: Carlos Francisco Sampaio Bonafe / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T01:21:44Z (GMT). No. of bitstreams: 1 Santos_JoseLuisdaRocha_D.pdf: 2961900 bytes, checksum: 8d0413331d106601d34efccd4f8e6bb7 (MD5) Previous issue date: 2008 / Resumo: Os vírus são estruturas auto-associaveis muito eficientes, porém, pouco é conhecido sobre a termodinâmica que governa essa associação dirigida. Em altos níveis de pressão ocorre dissociação do TMV ou desnaturação quando a pressão é combinada com uréia. Para um melhor entendimento de tais processos nós calculamos os parâmetros termodinâmicos aparentes de dissociação e desnaturação do TMV, assumindo uma condição de estado estacionário "steady-state condition". Esses processos podem ser monitorados pela diminuição de espalhamento de luz da solução virai devido ao processo de dissociação e pelo desvio para o vermelho do espectro de emissão de fluorescência, que ocorre com o processo de desnaturação. Nós determinamos a estequiometria aparente de uréia considerando a reação de equilíbrio de dissociação e desnaturação das subunidades do TMV, as quais forneceram, respectivamente, 1,53 e 11,1 mols de uréia/mol de subunidade de TMV. As condições de dissociação e desnaturação foram atingidas em uma via próxima da reversível, permitindo a determinação de parâmetros termodinâmicos. Analises de gel filtração em HPLC, microscopia eletrônica e dicroísmo circular confirmaram os processos de dissociação e desnaturação. Os cálculos das estequiometrias aparentes de uréia de vários vírus baseados em resultados espectroscópicos de artigos recentes mostraram que os processos de dissociação e desnaturação seguem uma estequiometria similar, sugerindo uma interação similar uréia-vírus entre estes sistemas. A desnaturação do TMV utilizando GndHCl foi mais eficiente que a uréia, apresentando estequiometria de 7,50 mols de GndHCl /mol de subunidade de TMV / Abstract: Viruses are very efficient self-assembly structures, but little is understood about the thermodynamics governing this directed assembly. At higher levels of pressure occurs dissociation of TMV or when pressure is combined with urea, denaturation occurs. For a better understanding of such processes, we investigated the apparent thermodynamic parameters of dissociation and denaturation by assuming a steady-state condition. These processes can be measured considering the decrease of light scattering of a viral solution due to the dissociation process, and the red shift of the fluorescence emission spectra, that occurs with the denaturation process. We determined the urea stoicbiometry considering the equilibrium reaction of TMV dissociation and subunit denaturation, which furnished, respectively, 1.53 and 11.1 mois of urea/mol of TMV subunit. The denaturation and dissociation conditions were arrived in a near reversible pathway, allowing the determination of thermodynamic parameters. Gel filtration HPLC, electron microscopy and circular dichroism confirmed the dissociation and denaturation processes. The calculation of apparent urea stoicbiometry of several other viruses based on spectroscopic results from earlier papers showed that dissociation and denaturation processes follow similar stoichiometrics, suggesting a similar virus-urea interaction among these systems. The TMV denaturation was more efficient in the presence of GndHCl, with stoichiometry of 7.50 mois of GndHCl /mol of TMV subunit / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Infectious diseases remain a significant cause of human deaths, as approximately 15 million deaths were attributed to infectious diseases in 2010 (Dye, 2014). One such disease is rabies, which causes around 59,000 human deaths worldwide annually according to some estimates (Kessels et al., 2017). However, 95% of human deaths attributed to rabies occur in Asia and Africa (Singh et al., 2017). Rabies is preventable, yet it is still a major concern in developing, low-income countries that lack access to the medical care necessary to combat it (Hampson et al., 2015). Alternative techniques for low-cost vaccine production have the potential to resolve this issue. This research investigates the use of recombinant DNA techniques and plant biotechnology to produce a more cost-effective vaccine for rabies. Gene sequences from the rabies glycoprotein were inserted at the end of the coat protein portion of the Tobacco Mosaic Virus (TMV) genome. Plants were then infected with this recombinant virus, with hopes that TMV particles would assemble with proteins produced from the inserted glycoprotein sequence fused to the TMV coat protein. Results thus far suggest some of the sequences could be producing recombinant TMV particles, although issues involving successful extraction and reversion to wild type are still a challenge. Additionally, other research suggests that this is an effective method for vaccine development in general and for rabies.

Double infection by cowpea strains of southern bean mosaic virus (CP-SBMV) and of tobacco mosaic virus (CP-TMV) caused additive growth reductions in California blackeye cowpea. Plant height, weight and numbers of seed and pods were significantly reduced by double infection and by CP-TMV single infection compared to healthy and CP-SBMV-sing 1 y infected plants. Singly and doubly inoculated California blackeye cowpea plants developed CP-SBMV symptoms on the primary leaves, but CP-SBMV symptoms in doubly infected trifoliates were masked by CP-TMV symptoms. CP-TMV symptoms did not mask CP-SBMV symptoms in systemically infected trifoliate leaves of another cowpea variety, V45-Bots. CP-TMV infection conditioned systemic infection of V45~Bots by CP-SBMV, as indicated by infectivity,serology and analytical sucrose density gradient centrifugation. CP-TMV also induced susceptibility of Pinto to infection by CP-SBMV, as ascertained by infectivity, immunodiffusion and electron microscopy. Analytical sucrose density gradient centrifugation measurements demonstrated that in doubly inoculated primary leaves of California blackeye cowpea CP-SBMV and CP-TMV were synthesized less than in the same leaves singly inoculated. CP-SBMV synthesis in trifoliate leaves, following simultaneous inoculations of primary leaves, was enhanced 5 times that in singly infected trifoliate leaves, whereas CP-TMV synthesis was not greatly affected. When CP-TMV preceded CP-SBMV in the primary leaves by 2k and 72 hr CP-SBMV synthesis was enhanced more in trifoliate leaves that were undifferentiated at the time of inoculation than in those of plants simultaneously inoculated. When CP-TMV preceded CP-SBMV into preformed 3rd trifoliate leaves by 22 hr, the ratio of CP-SBMV concentration in doubly infected tissue to that in singly infected tissue was 2.7 versus 1.9 when both viruses arrived simultaneously at these leaves. When either virus preceded the other by 72 hr into preformed 3rd trifoliate leaves the synthesis of the challenging virus was greatly retarded. CP-SBMV synthesis was also enhanced by CP-TMV infection under differential temperature synchronous system of infection. Although virions of both viruses were detected in the same cell no genomic masking was detected by infectivity neutralization test. It is theorized that CP-TMV infection predisposes the host cells to infection by CP-SBMV and thus the enhanced synthesis of CP-SBMV. The effect of CP-TMV infection on CP-SBMV synthesis in cowpea seems to be a physiological one. CP-SBMV, but not CP-TMV, was transmitted through planted seed and decontaminated embryos of California blackeye cowpea. Buffer extracts made from decontaminated embryos also were infectious for CP-SBMV. Seed coats contained both viruses. Double infection of California blackeye cowpea decreased seed transmission of CP-SBMV from 13-5 to 7.6%. Buffer extracts of healthy seed were inhibitory to infectivity of both viruses. Germination of seed reduced infectivity of CP-SBMV in the seed coats, but not of CP-TMV. It is also concluded that seed transmission of CP-SBMV is a result of embryo infection rather than contamination with virus in the seed coats. / Land and Food Systems, Faculty of / Graduate