@phdthesis{18766, abstract = {Poxviruses are large pleomorphic double-stranded DNA viruses that include well known members such as variola virus, the causative agent of smallpox, Mpox virus, as well as Vaccinia virus (VACV), which serves as a vaccination strain for formerly mentioned viruses. VACV is a valuable model for studying large pleomorphic DNA viruses in general and poxviruses specifically, as many features, such as core morphology and structural proteins, are well conserved within this family. Despite decades of research, our understanding of the structural components and proteins that comprise the poxvirus core in mature virions remains limited. Although major core proteins were identified via indirect experimental evidence, the core's complexity, with its large size, structure and number of involved proteins, has hindered efforts to achieve high-resolution insights and to define the roles of the individual proteins. The specific protein composition of the core's individual layers, including the palisade layer and the inner core wall, has remained unclear. In this study, we have merged multiple approaches, including single particle cryo electron microscopy of purified virus cores, cryo-electron tomography and subtomogram averaging of mature virions and molecular modeling to elucidate the structural determinants of the VACV core. Due to the lack of experimentally derived structures, either in situ or reconstituted in vitro, we used Alphafold to predict models of the putative major core protein candidates, A10, 23k, A3, A4, and L4. Our results show that the VACV core is composed of several layers with varying local symmetries, forming more intricate interactions than observed previously. This allowed us to identify several molecular building blocks forming the viral core lattice. In particular, we identified trimers of protein A10 as a major core structure that forms the palisade layer of the viral core. Additionally, we revealed that six petals of a flower shaped core pore within the core wall are composed of A10 trimers. Furthermore, we obtained a cryo-EM density for the inner core wall that could potentially accommodate an A3 dimer. Integrating descriptions of protein interactions from previous studies enabled us to provide a detailed structural model of the poxvirus core wall, and our findings indicate that the interactions within A10 trimers are likely consistent across orthopox- and parapoxviruses. This combined application of cryo-SPA and cryo-ET can help overcome obstacles in studying complex virus structures in the future, including their key assembly proteins, interactions, and the formation into a core lattice. Our work provides important fundamental new insights into poxvirus core architecture, also considering the recent re-emergence of poxviruses.}, author = {Datler, Julia}, isbn = {978-3-99078-049-7}, issn = {2663-337X}, keywords = {cryo-EM, cryo-ET, cryo-SPA, Structural Virology, Poxvirus, Vaccinia Virus, Structural Biology}, pages = {106}, publisher = {Institute of Science and Technology Austria}, title = {{Elucidating the structural determinants of the poxvirus core using multi-modal cryo-EM}}, doi = {10.15479/at:ista:18766}, year = {2024}, } @phdthesis{18443, abstract = {In [KW06] Kapustin and Witten conjectured that there is a mirror symmetry relation between the hyperkähler structures on certain Higgs bundle moduli spaces. As a consequence, they conjecture an equivalence between categories of BBB and BAA-branes. At the classical level, this mirror symmetry is given by T-duality between semi-flat hyperkähler structures on algebraic integrable systems. In this thesis, we investigate the T-duality relation between hyperkähler structures and the corresponding branes on affine torus bundles. We use the techniques of generalized geometry to show that semi-flat hyperkähler structures are T-dual on algebraic integrable systems. We also describe T-duality for generalized branes. Motivated by Fourier-Mukai transform we upgrade the T-duality between generalized branes to T-duality of submanifolds endowed with U(1)-bundles and connections. This T-duality in the appropriate context specializes to T-duality between BBB and BAA-branes. }, author = {Sisak, Maria A}, issn = {2663-337X}, keywords = {hyperkaehler geometry, branes, mirror symmetry, T-duality}, pages = {178}, publisher = {Institute of Science and Technology Austria}, title = {{T-dual branes on hyperkähler manifolds}}, doi = {10.15479/at:ista:18443}, year = {2024}, } @phdthesis{12491, abstract = {The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate. Despite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment. In this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET). To this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants. In order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness. High-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment. In summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions.}, author = {Zens, Bettina}, isbn = {978-3-99078-027-5}, issn = {2663-337X}, keywords = {cryo-EM, cryo-ET, FIB milling, method development, FIBSEM, extracellular matrix, ECM, cell-derived matrices, CDMs, cell culture, high pressure freezing, HPF, structural biology, tomography, collagen}, pages = {187}, publisher = {Institute of Science and Technology Austria}, title = {{Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography}}, doi = {10.15479/at:ista:12491}, year = {2023}, } @article{22540, abstract = {The reliable partitioning of the terrestrial latent heat flux into evaporation (E) and transpiration (T) is important for linking carbon and water cycles and for better understanding ecosystem functioning at local, regional and global scales. Previous research revealed that the transpiration-to-evapotranspiration ratio (T/ET) is well constrained across ecosystems and is nearly independent of vegetation characteristics and climate. Here we investigated the reasons for such a global constancy in present-day T/ET by jointly analysing observations and process-based model simulations. Using this framework, we also quantified how the ratio T/ET could be influenced by changing climate. For present conditions, we found that the various components of land surface evaporation (bare soil evaporation, below canopy soil evaporation, evaporation from interception), and their respective ratios to plant transpiration, depend largely on local climate and equilibrium vegetation properties. The systematic covariation between local vegetation characteristics and climate, resulted in a globally constrained value of T/ET = ∼70 ± 9% for undisturbed ecosystems, nearly independent of specific climate and vegetation attributes. Moreover, changes in precipitation amounts and patterns, increasing air temperatures, atmospheric CO2 concentration, and specific leaf area (the ratio of leaf area per leaf mass) was found to affect T/ET in various manners. However, even extreme changes in the aforementioned factors did not significantly modify T/ET.}, author = {Paschalis, Athanasios and Fatichi, Simone and Pappas, Christoforos and Or, Dani}, issn = {1748-9326}, journal = {Environmental Research Letters}, keywords = {T/ET, Evapotranspiration partitioning, Ecohydrology, Modelling, Climate change}, number = {10}, publisher = {IOP Publishing }, title = {{Covariation of vegetation and climate constrains present and future T/ET variability}}, doi = {10.1088/1748-9326/aae267}, volume = {13}, year = {2018}, }