In this preclinical two-dose mucosal immunization study, using a combination of S1 spike and nucleocapsid proteins with cationic (N3)/or anionic (L3) lipids were investigated using an intranasal delivery route. The study showed that nasal administration of low amounts of antigens/adjuvants induced a primary and secondary immune response in systemic IgG, mIL-5, and IFN-gamma secreting T lymphocytes, as well as humoral IgA in nasal and intestinal mucosal compartments. It is believed that recipients will benefit from receiving a combination of viral antigens in promoting a border immune response against present and evolving contagious viruses. Lipid adjuvants demonstrated an enhanced response in the vaccine effect. This was seen in the significant immunogenicity effect when using the cationic lipid N3. Unlike L3, which showed a recognizable effect when administrated at a slightly higher concentration. Moreover, the findings of the study proved the efficiency of an intranasally mucosal immunization strategy, which can be less painful and more effective in enhancing the respiratory tract immunity against respiratory infectious diseases.

This structural study exploits the possibility to use modular protein deuteration to facilitate the study of ubiquitin signalling, transfer, and modification. A protein conjugation reaction is used to combine protonated E2 enzyme with deuterated ubiquitin for small angle X-ray and neutron scattering with neutron contrast variation. The combined biomolecules stay as a monodisperse system during data collection in both protonated and deuterated buffers indicating long stability of the E2-Ub conjugate. With multiphase ab initio shape restoration and rigid body modelling, we reconstructed the shape of a E2-Ub-conjugated complex of UBE2D1 linked to ubiquitin via an isopeptide bond. Solution X-ray and neutron scattering data for this E2-Ub conjugate in the absence of E3 jointly indicate an ensemble of open and backbent states, with a preference for the latter in solution. The approach of combining protonated and labelled proteins can be used for solution studies to assess localization and movement of ubiquitin and could be widely applied to modular Ub systems in general.

This thesis uses small angle X-ray and neutron scattering (SAXS/SANS) to gain structural and functional insight into the molecular regulation of critical life processes in prokaryotic and eukaryotic species. The presented studies highlight the strength of combining low-resolution structure determination with biophysical and in silico modelling methods to extensively characterize proteins and their interactions.  

DNA-binding: MexR protein belongs to the family of bacterial transcription regulators and control the expression of multidrug efflux pumps in Pseudomonas Aeruginosa by binding to a DNA region of the operator. SAXS/SANS data supported by MD (Molecular Dynamics) simulations demonstrated that the MexR dimer in solution undergoes a DNA-binding conformational selection mechanism. To gain a better understanding about the system, a low-resolution structural model was resolved in order to assess protein binding to the entire operator region comprising of two closely located DNA recognition sites. The study demonstrates that the use of scattering techniques to investigate similar systems is straightforward and provides knowledge of relevance for clinical understanding and future drug design.  

Viral host factors: Picornaviruses represent a large family of small RNA viruses that are responsible for a range of diseases in humans and animals. Recently a non-essential human phospholipase PLAAT3 was identified as a key host factor for some picornaviruses. Several picornaviruses representing different branches of the picornaviral phylogenetic tree contain a type of 2A protein in their genome that share a conserved H-box/NC motif with PLAAT3. To understand the role of these 2A proteins in the viral life cycle and to map their plasticity, high resolution techniques were complemented with SAXS to evaluate the structural rearrangements and flexibility.  

Ubiquitination: In eukaryotes, ubiquitination is a fundamental posttranslational modification, where a small protein ubiquitin is covalently attached to a target protein via sophisticated multienzyme process. SANS can be used to study this mechanism in solution by modular deuteration of ubiquitin complexes. To explore this possibility further, an E2 conjugating enzyme was attached to a deuterated ubiquitin via an isopeptide bond, and a neutron contrast variation experiment was performed. To investigate the flexibility of the E2~Ub conjugate, a multi-state modelling approach was employed to sample its conformational landscape.  

SANS methods in protein science: A final methods paper outlines and details the experimental requirements, procedures and pre-studies that need to be considered to optimise a successful experimental approach for SANS with contrast variation on biomolecular complexes and assemblies in solution. 

In this work, we used Small-angle X-ray and neutron scattering (SAS) to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa (P.aeruginosa) transcriptional regulator MexR, a member of the MarR family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux-proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterisation of both free and DNA-bound MarR proteins. Here, we show how combined neutron and X-ray small-angle scattering (SAS) of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.

Small angle scattering affords an approach to evaluate the structure of dilute populations of macromolecules in solution where the measured scattering intensities relate to the distribution of scattering-pair distances within each macromolecule. When small angle neutron scattering (SANS) with contrast variation is employed, additional structural information can be obtained regarding the internal organization of biomacromolecule complexes and assemblies. The technique allows for the components of assemblies to be selectively ‘matched in’ and ‘matched out’ of the scattering profiles due to the different ways the isotopes of hydrogen—protium 1H, and deuterium 2H (or D)—scatter neutrons. The isotopic substitution of 1H for D in the sample enables the controlled variation of the scattering contrasts. A contrast variation experiment requires trade-offs between neutron beam intensity, q-range, wavelength and q-resolution, isotopic labelling levels, sample concentration and path-length, and measurement times. Navigating these competing aspects to find an optimal combination is a daunting task. Here we provide an overview of how to calculate the neutron scattering contrasts of dilute biological macromolecule samples prior to an experiment and how this then informs the approach to configuring SANS instruments and the measurement of a contrast variation series dataset.

Model organisms such as Drosophila melanogaster have been key tools for advancing our fundamental and applied knowledge in biological and biomedical sciences. However, model organisms have become intertwined with the idea of controlled and stable laboratory environments, and their natural history has been overlooked. In holometabolous insects, lack of natural history information on larval ecology has precluded major advances in the field of developmental ecology, especially in terms of manipulations of population density early in life (i.e., larval density). This is because of relativistic and to some extent, arbitrary methodologies employed to manipulate larval densities in laboratory studies. As a result, these methodologies render comparisons between species impossible, precluding our understanding of macroevolutionary responses to population densities during development that can be derived from comparative studies. We recently proposed a new conceptual framework to address this issue, and here, we provide the first natural history investigation of Drosophila melanogaster larval density under such framework. First, we characterized the distribution of larval densities in a wild population of D. melanogaster using rotting apples as breeding substrate in a suburban area in Sweden. Next, we compiled the commonly used methodologies for manipulating larval densities in laboratory studies from the literature and found that the majority of laboratory studies identified did not manipulate larval densities below or above the densities observed in nature, suggesting that we have yet to study true life history and physiological responses to low and high population densities during D. melanogaster development. This is, to our knowledge, the first direct natural history account of larval density in nature for this model organism. Our study paves the way for a more integrated view of organismal biology which re-incorporates natural history of model organisms into hypothesis-driven research in developmental ecology.

Herbivorous insects such as butterflies and moths are essential to natural and agricultural systems due to pollination and pest outbreaks. However, our knowledge of butterflies and moths nutrition is fragmented and limited to few common, charismatic, or problematic species. This gap precludes our complete understanding of herbivorous insects natural history, physiological and behavioral adaptations that drive how species interact with their environment, the consequences of habitat fragmentation and climate change to invertebrate biodiversity, and pest outbreak dynamics. Here, we first report a population of the Buff-tip moth Phalera bucephala (Lepidoptera: Notodontidae) feeding on a previously unknown family of host plants, the mountain currant Ribes alpinum (Saxifragales: Grossulariaceae). This is the first report of a Notodontid moth feeding on Grossulariaceae hosts. Using no-choice and choice assays, we showed that P. bucephala has strong foraging preferences for a previously unknown hosts, the R. alpinum but also, although to a smaller extent, R. uva-crispa compared with a previously known host (the Norway maple Acer sp.). These findings demonstrate that P. bucephala feed on-and show strong preference for Grossulariaceae host plants, indicating flexible physiological mechanisms to accommodate hosts plants from various families. This makes this species a potential model organism to study the behavioral and physiological mechanisms underpinning insect-plant interactions and diet breadth evolution. We discuss the broad ecological implications of these observations to the biology of the species, the potential negative effects of interspecific competition with endemic specialist moths, and highlight questions for future research.