Understanding the Speciation of Ruthenium Arene Complexes as Precursors to Unnatural Enzyme Cofactors

Ruthenium arene complexes have been extensively explored as metallo-pharmaceuticals and as small molecule catalysts. Exploring the overlap between these areas, this thesis describes a body of work aimed at quantitatively understanding the biological speciation and catalytic behaviour of ruthenium arene complexes when exposed to the many potential Lewis basic ligands provided by protein scaffolds. Combining nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), X-ray crystallography and other biophysical characterisation techniques, the speciation of ruthenium arene bipyridine complexes with small molecule amino acids, short peptides and whole proteins has been monitored. A 19F NMR spectroscopic method was developed to quantitively trace the preferred amino acid binding partners of ruthenium complexes coordinated to fluorinated ligands. Liquid chromatography mass spectrometry (LC-MS) was used to explore the ligand exchange behaviour between ruthenium complexes and protein scaffolds, particularly variants of the small proteins ubiquitin and cytochrome b562. Additionally tandem MS/MS experiments were used to determine the final protein binding sites of non-fluorinated ruthenium fragments coordinated to proteins. The resulting deep understanding of how ruthenium arene complexes coordinate to specific proteins was used to develop artificial metalloenzymes (ArMs) with direct protein-metal coordination. The catalytic capabilities of these hybrid systems was then explored. ArMs are synthetic biocatalysts that result from either the combination of an artificial metallo-cofactor being introduced into a protein scaffold or a natural metalloprotein being evolved to perform catalytic reactivity. Taking inspiration from naturally occurring metalloproteins, this research showed that it was possible to form ArMs via a ligand exchange process between a ruthenium arene precursor complex and a protein scaffold, resulting in the precursor complex being activated towards catalysis. Direct protein – metal coordination enables the protein to impart both an electronic and steric contribution to catalysis and attenuate reactivity at the metal centre in ways that have not been previously studied. The four helical bundle protein cytochrome b562 was selected as the protein scaffold for ArM development due to its dynamic structure and nascent haem binding site, which in the absence of haem provides a hydrophobic pocket capable of accommodating a ruthenium cofactor and catalytic substrate. Cytochrome b562 – ruthenium hybrids (with direct coordination) were identified that have catalytic transfer hydrogenation activity greater than a known dimeric catalyst. This demonstrates an exciting starting point to explore the evolutionary potential of these ArMs through directed evolution, hopefully enhancing catalytic activity

Thermal X-rays from Millisecond Pulsars: Constraining the Fundamental Properties of Neutron Stars

Abridged) We model the X-ray properties of millisecond pulsars (MSPs) by considering hot spot emission from a weakly magnetized rotating neutron star (NS) covered by an optically-thick hydrogen atmosphere. We investigate the limitations of using the thermal X-ray pulse profiles of MSPs to constrain the mass-to-radius ($M/R$) ratio of the underlying NS. The accuracy is strongly dependent on the viewing angle and magnetic inclination. For certain systems, the accuracy is ultimately limited only by photon statistics implying that future X-ray observatories could, in principle, achieve constraints on $M/R$ and hence the NS equation of state to better than $\sim$5%. We demonstrate that valuable information regarding the basic properties of the NS can be extracted even from X-ray data of fairly limited photon statistics through modeling of archival spectroscopic and timing observations of the nearby isolated PSRs J0030+0451 and J2124--3358. The X-ray emission from these pulsars is consistent with the presence of a hydrogen atmosphere and a dipolar magnetic field configuration, in agreement with previous findings for PSR J0437--4715. For both MSPs, the favorable geometry allows us to place interesting limits on the allowed $M/R$ of NSs. Assuming 1.4 M$_{\odot}$, the stellar radius is constrained to be $R > 9.4$ km and $R > 7.8$ km (68% confidence) for PSRs J0030+0451 and J2124--3358, respectively. We explore the prospects of using future observatories such as \textit{Constellation-X} and \textit{XEUS} to conduct blind X-ray timing searches for MSPs not detectable at radio wavelengths due to unfavorable viewing geometry. Using the observational constraints on the pulsar obliquities we are also able to place strong constraints on the magnetic field evolution model proposed by Ruderman.Comment: 9 pages, 7 figures, published in the Astrophysical Journal (Volume 689, Issue 1, pp. 407-415

Kaip meniniai tyrimai gali padėti atsakyti į auditorijų įsitraukimo klausimus?

[full article and abstract in Lithuanian; abstract in English] There is an increasing interest in audience engagement and the possibilities of its research in contemporary performance practice. One of the possibilities that can be used in conducting such a study is a practice-led type of research, commonly known as artistic research. The distinguishing quality of this type of research, which has been finding its place in academia throughout the last few decades, is that it uses artistic practice to generate new knowl­edge. This article analyzes the possibilities of such research from the point of view of the aesthetics of performativity, which focuses on works that explore audience engagement rather than representation. While analyzing examples of performance and theater art works, it tackles the issue of how artistic research can assist in answering audience engagement questions.[straipsnis ir santrauka lietuvių kalba; santrauka anglų kalba] Šiuolaikiniuose scenos menuose pastebimas augantis susidomėjimas auditorijų įsitraukimo mechanizmais, siekiama juos permąstyti ir tirti. Vienas iš tyrimo būdų yra per pastaruosius keletą dešimtmečių vis didesnį pripažinimą įgaunanti meninių tyrimų sritis, kurioje kūrybinė praktika pasitelkiama kaip įrankis tyrimui vykdyti. Šiame straipsnyje į meninius tyrimus žvelgiama iš scenos menų perspektyvos, kur jie siejami su performatyvumo estetikos, sureikšminančios ne reprezentaciją, o gyvą dialogą tarp atlikėjo ir žiūrovo, atsiradimu. Pateikiant praktinius pavyzdžius, mėginama atsakyti į klausimą – kaip meniniai tyrimai gali padėti atsakyti į auditorijų įsitraukimo klausimus

Responsive biomaterials:advances in materials based on shape-memory polymers

Shape-memory polymers (SMPs) are morphologically responsive materials with potential for a variety of biomedical applications, particularly as devices for minimally invasive surgery and the delivery of therapeutics and cells for tissue engineering. A brief introduction to SMPs is followed by a discussion of the current progress toward the development of SMP-based biomaterials for clinically relevant biomedical applications

An improved collision efficiency model for particle aggregation

A generalized geometric model is presented which describes the collision efficiency factor of aggregation (the probability of a binary particle or aggregate collision resulting in adhesion) for systems comprised of two oppositely charged species. Application of the general model to specific systems requires calculation of the area of each species available for collision with a second species. This is in contrast to previous models developed for polymer-particle flocculation that are based on the fractional surface coverage of adsorbed polymer. The difference between these approaches is suggested as an explanation for previously observed discrepancies between theory and observation. In the current work the specific case of oppositely charged nondeformable spherical particles (heteroaggregation) is quantitatively addressed. The optimum concentration of oppositely charged particles for rapid aggregation (maximum collision efficiency) as a function of relative particle size is calculated and an excellent correlation is found with data taken from literature

Combining experiment and energy landscapes to explore anaerobic heme breakdown in multifunctional hemoproteins

To survive, many pathogens extract heme from their host organism and break down the porphyrin scaffold to sequester the Fe2+ ion via a heme oxygenase. Recent studies have revealed that certain pathogens can anaerobically degrade heme. Our own research has shown that one such pathway proceeds via NADH-dependent heme degradation, which has been identified in a family of hemoproteins from a range of bacteria. HemS, from Yersinia enterocolitica, is the main focus of this work, along with HmuS (Yersinia pestis), ChuS (Escherichia coli) and ShuS (Shigella dysenteriae). We combine experiments, Energy Landscape Theory, and a bioinformatic investigation to place these homologues within a wider phylogenetic context. A subset of these hemoproteins are known to bind certain DNA promoter regions, suggesting not only that they can catalytically degrade heme, but that they are also involved in transcriptional modulation responding to heme flux. Many of the bacterial species responsible for these hemoproteins (including those that produce HemS, ChuS and ShuS) are known to specifically target oxygen-depleted regions of the gastrointestinal tract. A deeper understanding of anaerobic heme breakdown processes exploited by these pathogens could therefore prove useful in the development of future strategies for disease prevention