Biophysical studies to elucidate structure-activity relationships in β-defensins

β-defensins are a class of mammalian defence peptides with therapeutic potential because of their ability to kill bacteria and attract host immune cells. In order to realise this potential, it is necessary to understand how the functions of these peptides are related to their structures. This thesis presents biophysical analysis of β- defensins and related peptides in conjunction with biological assays. These studies provide new insights into the structure-activity relationships of β-defensins. Ion mobility-mass spectrometry (IM-MS) is used throughout this thesis to probe the tertiary structure of peptides in vacuo and, by inference, make conclusions about their conformations in solution prior to ionisation. Where appropriate, IM-MS is complemented by other techniques, including high performance liquid chromatography and circular dichroism spectroscopy. First, the importance of a C-terminal cysteine residue within the murine β-defensin Defb14 is investigated. The functional and structural implications of chemically modifying the cysteine residue are examined. Second, the N-terminal region of Defb14 is modified by the substitution and deletion of amino acids. Again, the effects on biological activity and structure are discussed. Finally, the functional and structural overlap of β-defensins with another family of proteins – the chemokines – is considered. The oligomerisation of β-defensins and their interaction with glycosaminoglycans is of particular interest: structural data for human β-defensins 2 and 3 in the absence and presence of polysaccharides are presented

Peptide Fragments of a β-Defensin Derivative with Potent Bactericidal Activity

β-Defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine β-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this β-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide

Automatic coronary calcium scoring in chest CT using a deep neural network in direct comparison with non-contrast cardiac CT:A validation study

Purpose: To evaluate deep-learning based calcium quantification on Chest CT scans compared with manual evaluation, and to enable interpretation in terms of the traditional Agatston score on dedicated Cardiac CT. Methods: Automated calcium quantification was performed using a combination of deep-learning convolution neural networks with a ResNet-architecture for image features and a fully connected neural network for spatial coordinate features. Calcifications were identified automatically, after which the algorithm automatically excluded all non-coronary calcifications using coronary probability maps and aortic segmentation. The algorithm was first trained on cardiac-CTs and refined on non-triggered chest-CTs. This study used on 95 patients (cohort 1), who underwent both dedicated calcium scoring and chest-CT acquisitions using the Agatston score as reference standard and 168 patients (cohort 2) who underwent chest-CT only using qualitative expert assessment for external validation. Results from the deep-learning model were compared to Agatston-scores(cardiac-CTs) and manually determined calcium volumes(chest-CTs) and risk classifications. Results: In cohort 1, the Agatston score and AI determined calcium volume shows high correlation with a correlation coefficient of 0.921(p < 0.001) and R-2 of 0.91. According to the Agatston categories, a total of 67(70 %) were correctly classified with a sensitivity of 91 % and specificity of 92 % in detecting presence of coronary calcifications. Manual determined calcium volume on chest-CT showed excellent correlation with the AI volumes with a correlation coefficient of 0.923(p < 0.001) and R-2 of 0.96, no significant difference was found (p = 0.247). According to qualitative risk classifications in cohort 2, 138(82 %) cases were correctly classified with a k-coefficient of 0.74, representing good agreement. All wrongly classified scans (30(18 %)) were attributed to an adjacent category. Conclusion: Artificial intelligence based calcium quantification on chest-CTs shows good correlation compared to reference standards. Fully automating this process may reduce evaluation time and potentially optimize clinical calcium scoring without additional acquisitions

STREGA: STRucture and Evolution of the GAlaxy - I : Survey overview and first results

STREGA (STRucture and Evolution of the GAlaxy) is a guaranteed time survey being performed at the VST (the ESO Very Large Telescope Survey Telescope) to map about 150 square degrees in the Galactic halo, in order to constrain the mechanisms of galactic formation and evolution. The survey is built as a 5 yr project, organized in two parts: a core programme to explore the surrounding regions of selected stellar systems and a second complementary part to map the southern portion of the Fornax orbit and extend the observations of the core programme. The adopted stellar tracers are mainly variable stars (RR Lyraes and long-period variables) and main-sequence turn-off stars for which observations in the g, r, i bands are obtained. We present an overview of the survey and some preliminary results for three observing runs that have been completed. For the region centred on ω Cen (37 deg^2), covering about three tidal radii, we also discuss the detected stellar density radial profile and angular distribution, leading to the identification of extratidal cluster stars. We also conclude that the cluster tidal radius is about 1.2 deg, in agreement with values in the literature based on the Wilson model.Peer reviewedFinal Accepted Versio

First Observation of the Rare Decay Mode K-long -> e+ e-

In an experiment designed to search for and study very rare two-body decay modes of the K-long, we have observed four examples of the decay K-long -> e+ e-, where the expected background is 0.17+-0.10 events. This observation translates into a branching fraction of 8.7^{+5.7}_{-4.1} X 10^{-12}, consistent with recent theoretical predictions. This result represents by far the smallest branching fraction yet measured in particle physics.Comment: 9 pages, 3 figure

Inclusive Search for Anomalous Production of High-pT Like-Sign Lepton Pairs in Proton-Antiproton Collisions at sqrt{s}=1.8 TeV

We report on a search for anomalous production of events with at least two charged, isolated, like-sign leptons with pT > 11 GeV/c using a 107 pb^-1 sample of 1.8 TeV ppbar collisions collected by the CDF detector. We define a signal region containing low background from Standard Model processes. To avoid bias, we fix the final cuts before examining the event yield in the signal region using control regions to test the Monte Carlo predictions. We observe no events in the signal region, consistent with an expectation of 0.63^(+0.84)_(-0.07) events. We present 95% confidence level limits on new physics processes in both a signature-based context as well as within a representative minimal supergravity (tanbeta = 3) model.Comment: 15 pages, 4 figures. Minor textual changes, cosmetic improvements to figures and updated and expanded reference

Measurement of the Lifetime Difference Between B_s Mass Eigenstates

We present measurements of the lifetimes and polarization amplitudes for B_s --> J/psi phi and B_d --> J/psi K*0 decays. Lifetimes of the heavy (H) and light (L) mass eigenstates in the B_s system are separately measured for the first time by determining the relative contributions of amplitudes with definite CP as a function of the decay time. Using 203 +/- 15 B_s decays, we obtain tau_L = (1.05 +{0.16}/-{0.13} +/- 0.02) ps and tau_H = (2.07 +{0.58}/-{0.46} +/- 0.03) ps. Expressed in terms of the difference DeltaGamma_s and average Gamma_s, of the decay rates of the two eigenstates, the results are DeltaGamma_s/Gamma_s = (65 +{25}/-{33} +/- 1)%, and DeltaGamma_s = (0.47 +{0.19}/-{0.24} +/- 0.01) inverse ps.Comment: 8 pages, 3 figures, 2 tables; as published in Physical Review Letters on 16 March 2005; revisions are for length and typesetting only, no changes in results or conclusion

Measurement of Lifetime and Decay-Width Difference in B0s -> J/psi phi Decays

We measure the mean lifetime, tau=2/(Gamma_L+Gamma_H), and the width difference, DeltaGamma=Gamma_L-Gamma_H, of the light and heavy mass eigenstates of the B0s meson, B0sL and B0sH, in B0s -> J/psi phi decays using 1.7 fb^-1 of data collected with the CDF II detector at the Fermilab Tevatron ppbar collider. Assuming CP conservation, a good approximation for the B0s system in the Standard Model, we obtain DeltaGamma = 0.076^+0.059_-0.063 (stat.) +- 0.006 (syst.) ps^-1 and tau = 1.52 +- 0.04 (stat.) +- 0.02 (syst.) ps, the most precise measurements to date. Our constraints on the weak phase and DeltaGamma are consistent with CP conservation. Dedicated to the memory of our dear friend and colleague, Michael P. Schmid