STRUCTURAL ANALYSIS OF RIBOSOME BINDING ELEMENTS OF THE 3´ UTR IN TWO POSITIVE-STRAND PLANT RNA VIRUSES

Turnip crinkle virus (TCV) and Pea enation mosaic virus (PEMV) are two positive (+)-strand RNA viruses that are used to investigate the regulation of translation and replication due to their small size and simple genomes. Both viruses contain cap-independent translation elements (CITEs) within their 3´ untranslated regions (UTRs) that fold into tRNA-shaped structures (TSS) according to nuclear magnetic resonance and small angle x-ray scattering analysis (TCV) and computational prediction (PEMV). Specifically, the TCV TSS can directly associate with ribosomes and participates in RNA-dependent RNA polymerase (RdRp) binding. The PEMV kissing-loop TSS (kl-TSS) can simultaneously bind to ribosomes and associate with the 5´ UTR of the viral genome. Mutational analysis and chemical structure probing methods provide great insight into the function and secondary structure of the two 3´ CITEs. However, lack of 3-D structural information has limited our understanding of their functional dynamics. Here, I report the folding dynamics for the TCV TSS using optical tweezers (OT), a single molecule technique. My study of the unfolding/folding pathways for the TCV TSS has provided an unexpected unfolding pathway, confirmed the presence of Ψ3 and hairpin elements, and suggested an interconnection between the hairpins and pseudoknots. In addition, this study has demonstrated the importance of the adjacent upstream adenylate-rich sequence for the formation of H4a/Ψ3 along with the contribution of magnesium to the stability of the TCV TSS. In my second project, I report on the structural analysis of the PEMV kl-TSS using NMR and SAXS. This study has re-confirmed the base-pair pattern for the PEMV kl-TSS and the proposed interaction of the PEMV kl-TSS with its interacting partner, hairpin 5H2. The molecular envelope of the kl-TSS built from SAXS analysis suggests the kl-TSS has two functional conformations, one of which has a different shape from the previously predicted tRNA-shaped form. Along with applying biophysical methods to study the structural folding dynamics of RNAs, I have also developed a technique that improves the production of large quantities of recombinant RNAs in vivo for NMR study. In this project, I report using the wild-type and mutant E.coli strains to produce cost-effective, site-specific labeled, recombinant RNAs. This technique was validated with four representative RNAs of different sizes and complexity to produce milligram amounts of RNAs. The benefit of using site-specific labeled RNAs made from E.coli was demonstrated with several NMR techniques.

The structural and mechanical integrity of historic wood

Little is known about historic wood as it ages naturally. Instead, most studies focus on biological decay, as it is often assumed that wood remains otherwise stable with age. This PhD project was organised by Historic Scotland and the University of Glasgow to investigate the natural chemical and physical aging of wood. The natural aging of wood was a concern for Historic Scotland as traditional timber replacement is the standard form of repair used in wooden cultural heritage; replacing rotten timber with new timber of the same species. The project was set up to look at what differences could exist both chemically and physically between old and new wood, which could put unforeseen stress on the joint between them. Through Historic Scotland it was possible to work with genuine historic wood from two species, Oak and Scots pine, both from the 1500’s, rather than relying on artificial aging. Artificial aging of wood is still a debated topic, with consideration given to whether it is truly mimicking the aging process or just damaging the wood cells. The chemical stability of wood was investigated using Fourier-transform infrared (FTIR) microscopy, as well as wet chemistry methods including a test for soluble sugars from the possible breakdown of the wood polymers. The physical properties assessed included using a tensile testing machine to uncover possible differences in mechanical properties. An environmental chamber was used to test the reaction to moisture of wood of different ages, as moisture is the most damaging aspect of the environment to wooden cultural objects. The project uncovered several differences, both physical and chemical, between the modern and historic wood which could affect the success of traditional ‘like for like’ repairs. Both oak and pine lost acetyl groups, over historic time, from their hemicellulose polymers. This chemical reaction releases acetic acid, which had no effect on the historic oak but was associated with reduced stiffness in historic pine, probably due to degradation of the hemicellulose polymers by acid hydrolysis. The stiffness of historic oak and pine was also reduced by decay. Visible pest decay led to loss of wood density but there was evidence that fungal decay, extending beyond what was visible, degraded the S2 layer of the pine cell walls, reducing the stiffness of the wood by depleting the cellulose microfibrils most aligned with the grain. Fungal decay of polysaccharides in pine wood left behind sugars that attracted increased levels of moisture. The degradation of essential polymers in the wood structure due to age had different impacts on the two species of wood, and raised questions concerning both the mechanism of aging of wood and the ways in which traditional repairs are implemented, especially in Scots pine. These repairs need to be done with more care and precision, especially in choosing new timber to match the old. Within this project a quantitative method of measuring the microfibril angle (MFA) of wood using polarised Fourier transform infrared (FTIR) microscopy has been developed, allowing the MFA of both new and historic pine to be measured. This provides some of the information needed for a more specific match when selecting replacement timbers for historic buildings.

A contribution towards the performance and structural understanding of copper and nickel salts as ammonia stores

The ongoing depletion of fossil fuels and the severe consequences of the greenhouse effect make the development of alternative energy systems crucially important. While hydrogen is, in principle, a promising alternative, releasing nothing but energy and pure water. Hydrogen storage is complicated and no completely viable technique has been proposed so far. This work is concerned with the study of one potential alternative to pure hydrogen: ammonia, and more specifically its storage in solids. Ammonia, NH3, can be regarded as a chemical hydrogen carrier with the advantages of strongly reduced flammability and explosiveness as compared to hydrogen. Furthermore, ammine metal salts presented here as promising ammonia stores easily store up to 50 wt.-% ammonia, giving them a volumetric energy density comparable to natural gas. The model system NiX2–NH3 ( X = Cl, Br, I) is studied thoroughly with respect to ammine salt formation, thermal decomposition, air stability and structural effects. The system CuX2–NH3 ( X = Cl, Br) has an adverse thermal decomposition behaviour, making it impractical for use as an ammonia store. This system is, however, most interesting from a structural point of view and some work concerning the study of the structural behaviour of this system is presented. Finally, close chemical relatives to the metal ammine halides, the metal ammine nitrates are studied. They exhibit interesting anion arrangements, which is an impressive showcase for the combination of diffraction and spectroscopic information. The characterisation techniques in this thesis range from powder diffraction over single crystal diffraction, spectroscopy, computational modelling, thermal analyses to gravimetric uptake experiments. Further highlights are the structure solutions and refinements from powder data of (NH4)2[NiCl4(H2O)(NH3)] and Ni(NH3)2(NO3)2, the combination of crystallographic and chemical information for the elucidation of the (NH4)2[NiCl4(H2O)(NH3)] formation reaction and the growth of single crystals under ammonia flow, a technique allowing the first documented successful growth and single crystal diffraction measurement for [Cu(NH3)6]Cl2.

THE IMPACT OF UPPER-LEVEL PROCESSES ON THE INTENSITY AND STRUCTURAL CHANGES OF HURRICANE SANDY (2012)

The first part of this study examines the relative roles of frontogenesis and tropopause undulation in determining the intensity and structural changes of Hurricane Sandy (2012) using a high-resolution cloud-resolving model. A 138-h simulation reproduces Sandy’s four distinct development stages: (i) rapid intensification, (ii) weakening, (iii) steady maximum surface wind but with large continued sea-level pressure (SLP) falls, and (iv) re-intensification. Results show typical correlations between intensity changes, sea-surface temperature and vertical wind shear during the first two stages. The large SLP falls during the last two stages are mostly caused by Sandy’s moving northward into lower-tropopause regions associated with an eastward-propagating midlatitude trough, where the associated lower-stratospheric warm air wraps into the storm and its surrounding areas. The steady maximum surface wind occurs because of the widespread SLP falls with weak pressure gradients lacking significant inward advection of absolute angular momentum (AAM). Meanwhile, there is a continuous frontogenesis in the outer region during the last three stages. Cyclonic inward advection of AAM along each frontal rainband accounts for the continued expansion of the tropical-storm-force wind and structural changes, while deep convection in the eyewall and merging of the final two survived frontal rainbands generate a spiraling jet in Sandy’s northwestern quadrant, leading to its re-intensification prior to landfall. The physical, kinematic and dynamic aspects of an upper-level outflow layer and its possible impact on the re-intensification of Sandy are examined in the second part of this study. Above the outflow layer isentropes are tilted downward with radius as a result of the development of deep convection and an approaching upper-level trough, causing weak subsidence. Its maximum outward radial velocity is located above the cloud top, so the outflow channel experiences cloud-induced long-wave cooling. Because Sandy has two distinct convective regions (an eyewall and a frontal rainband), it has multiple outflow layers, with the eyewall’s outflow layer located above that of the frontal rainband. During the re-intensification stage, the eyewall’s outflow layer interacts with a jet stream ahead of the upper-level trough axis. Because of the presence of inertial instability on the anticyclonic side of the jet stream and symmetric instability in the inner region of the outflow layer, Sandy’s secondary circulation intensifies. Its re-intensification ceases when these instabilities disappear. The relationship between the intensity of the secondary circulation and dynamic instabilities of the outflow layer suggests that the re-intensification occurs in response to these instabilities. Additionally, it is verified that the long-wave cooling in the outflow layer helps induce symmetric instability by reducing static stability.

Structural characterization and comparative analysis of human and piscine cartilage acidic protein (CRTAC1/CRTAC2)

Dissertação de mestrado, Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Improved mechanical stability of HKUST-1 in confined nanospace

One of the main concerns in the technological application of several metal–organic frameworks (MOFs) relates to their structural instability under pressure (after a conforming step). Here we report for the first time that mechanical instability can be highly improved via nucleation and growth of MOF nanocrystals in the confined nanospace of activated carbons.

Thermal decomposition of the synthetic hydrotalcite iowaite

The thermal stability and thermal decomposition pathways for synthetic iowaite have been determined using thermogravimetry in conjunction with evolved gas mass spectrometry. Chemical analysis showed the formula of the synthesised iowaite to be Mg6.27Fe1.73(Cl)1.07(OH)16(CO3)0.336.1H2O and X-ray diffraction confirms the layered structure. Dehydration of the iowaite occurred at 35 and 79°C. Dehydroxylation occurred at 254 and 291°C. Both steps were associated with the loss of CO2. Hydrogen chloride gas was evolved in two steps at 368 and 434°C. The products of the thermal decomposition were MgO and a spinel MgFe2O4. Experimentally it was found to be difficult to eliminate CO2 from inclusion in the interlayer during the synthesis of the iowaite compound and in this way the synthesised iowaite resembled the natural mineral.

Exploring first-year academic achievement through structural equation modelling

The purpose of this research was to develop and test a multicausal model of the individual characteristics associated with academic success in first-year Australian university students. This model comprised the constructs of: previous academic performance, achievement motivation, self-regulatory learning strategies, and personality traits, with end-of-semester grades the dependent variable of interest. The study involved the distribution of a questionnaire, which assessed motivation, self-regulatory learning strategies and personality traits, to 1193 students at the start of their first year at university. Students' academic records were accessed at the end of their first year of study to ascertain their first and second semester grades. This study established that previous high academic performance, use of self-regulatory learning strategies, and being introverted and agreeable, were indicators of academic success in the first semester of university study. Achievement motivation and the personality trait of conscientiousness were indirectly related to first semester grades, through the influence they had on the students' use of self-regulatory learning strategies. First semester grades were predictive of second semester grades. This research provides valuable information for both educators and students about the factors intrinsic to the individual that are associated with successful performance in the first year at university.