Method Development for the Detection and Identification of Pathophysiologically Relevant Electrophiles in Pollen

Pollen allergy is a complex, multicausal disease with rising prevalence across the world. Currently, pollen allergy is treated mainly by symptom management and allergen-specific immunotherapy. In order to develop new treatment options, a greater understanding of the underlying mechanism of the disease is needed. Based on disease hypotheses like the hapten theory and the danger model, electrophilic small molecules in pollen could play a part in the sensitisation process and the exacerbative nature of the disease. The research on small molecules in pollen is lagging behind in contrast to their protein counterparts, which is why the aim of this project was to develop a method to enable the detection and identification of electrophiles in pollen extracts. Method development in this work explored i) in situ detection and identification methods with liquid chromatography-mass spectrometry and ii) nucleophilic labelling with an affinity tag in order to facilitate a subsequent purification step, before investigating the potential of using a solid-supported nucleophilic probe. Finally, a probe was developed, consisting of a polystyrene solid support, a hyperacid-sensitive linker and a disulfide-protected cysteine that could act as a nucleophile to capture the electrophilic target molecules upon deprotection. The advantages of the probe are the following: i) the nucleophilic cysteine could be selectively deprotected, and a method was developed to quantify the released cysteine (8.65 ± 2.65 %) and therefore the amount of reactive sites on the resin; ii) the solid nature of the probe enabled a set- up in cartridges intended for solid-phase extraction, which allowed consecutive washes and reagent additions; iii) the hyperacid sensitive linker enabled the release of formed cysteine adducts after reaction; and finally, iv) due to the design of the probe, only mono-addition of cysteine was observed, except in cases where adducts could decompose. The probe was tested on model compounds, a model extract that was spiked with model compound and lastly, on diverse pollen extracts (Ambrosia psilostachya, Ambrosia artemisiifolia, Phleum pratense, Betula pendula, Urtica dioica, Corylus avellana). Both model compound and model extract experiments were successful; adduct formation was observed and the adducts were successfully isolated and characterised by nuclear magnetic resonance. However, due to the low abundance of electrophilic compounds in the extract, it unfortunately was not possible to isolate and characterise any compounds from pollen extracts, aside from two compounds isolated from a larger pollen extract experiment with Ambrosia psilostachya. The two isolated compounds were shown to be coumaroyl spermidine- like structures, however, their exact structure could not be determined

Non-invasive 19F NMR analysis of a protein-templated N- acylhydrazone dynamic combinatorial library. OBC 2018

Supplementary data to support the following publication (abstract): Dynamic combinatorial chemistry (DCC) is a powerful tool to identify ligands for biological targets. We used 19F NMR as an in situ, non-invasive technique for measuring the composition of a dynamic combinatorial library (DCL) of N-acylhydrazones (NAHs). An NAH DCL, constructed from a fluoro-aromatic aldehyde and a small set of hydrazides, was targetted at ecFabH, an essential enzyme in bacterial fatty acid biosynthesis. Our NMR analysis identified a tert-butyl NAH as the best binder which was confirmed by enzymatic assay.Ekstrom, Alexander; Campopiano, Dominic; Wang, Jue Theresa; Bella, Juraj. (2018). Non-invasive 19F NMR analysis of a protein-templated N- acylhydrazone dynamic combinatorial library. OBC 2018, [dataset]. University of Edinburgh. School of Chemistry. https://doi.org/10.7488/ds/2436

Measurements of ttˉt\bar{t} differential cross-sections of highly boosted top quarks decaying to all-hadronic final states in pppp collisions at s=13 \sqrt{s}=13\, TeV using the ATLAS detector

Measurements are made of differential cross-sections of highly boosted pair-produced top quarks as a function of top-quark and $t\bar{t}$ system kinematic observables using proton--proton collisions at a center-of-mass energy of $\sqrt{s} = 13$ TeV. The data set corresponds to an integrated luminosity of $36.1$ fb$^{-1}$, recorded in 2015 and 2016 with the ATLAS detector at the CERN Large Hadron Collider. Events with two large-radius jets in the final state, one with transverse momentum $p_{\rm T} > 500$ GeV and a second with $p_{\rm T}>350$ GeV, are used for the measurement. The top-quark candidates are separated from the multijet background using jet substructure information and association with a $b$-tagged jet. The measured spectra are corrected for detector effects to a particle-level fiducial phase space and a parton-level limited phase space, and are compared to several Monte Carlo simulations by means of calculated $\chi^2$ values. The cross-section for $t\bar{t}$ production in the fiducial phase-space region is $292 \pm 7 \ \rm{(stat)} \pm 76 \rm{(syst)}$ fb, to be compared to the theoretical prediction of $384 \pm 36$ fb

Measurements of ttˉt\bar{t} differential cross-sections of highly boosted top quarks decaying to all-hadronic final states in pppp collisions at s=13 \sqrt{s}=13\, TeV using the ATLAS detector

Measurements are made of differential cross-sections of highly boosted pair-produced top quarks as a function of top-quark and $t\bar{t}$ system kinematic observables using proton--proton collisions at a center-of-mass energy of $\sqrt{s} = 13$ TeV. The data set corresponds to an integrated luminosity of $36.1$ fb$^{-1}$, recorded in 2015 and 2016 with the ATLAS detector at the CERN Large Hadron Collider. Events with two large-radius jets in the final state, one with transverse momentum $p_{\rm T} > 500$ GeV and a second with $p_{\rm T}>350$ GeV, are used for the measurement. The top-quark candidates are separated from the multijet background using jet substructure information and association with a $b$-tagged jet. The measured spectra are corrected for detector effects to a particle-level fiducial phase space and a parton-level limited phase space, and are compared to several Monte Carlo simulations by means of calculated $\chi^2$ values. The cross-section for $t\bar{t}$ production in the fiducial phase-space region is $292 \pm 7 \ \rm{(stat)} \pm 76 \rm{(syst)}$ fb, to be compared to the theoretical prediction of $384 \pm 36$ fb