Low-Energy Electron Interactions with Radiosensitisers and Hydrated Biomolecular Clusters

Interactions of free electrons with kinetic energies between 0 eV and 100 eV with isolated biomolecules, (hydrated) radiosensitisers and doped neon clusters were studied, with focus on associative and dissociative electron attachment (AA and DEA, respectively) processes and electron ionisation. Radiosensitisers are applied in radiotherapy to enhance the ratio of damage to malignant compared to healthy cells. The principle of action on a physico-chemical stage is yet unknown and was studied within this thesis. Clusters are an intermediate state of matter between gaseous and solid state. In this thesis, cluster size dependencies and effects in doped clusters were studied. Two different mass spectrometry setups were used. Data from electron interactions with both gas-phase biomolecules and doped neon clusters was taken at the experimental apparatus in Innsbruck. A hemispherical electron monochromator enables high energy resolution measurements and is combined with a quadrupole mass filter. For the acquisition of hydrated radiosensitiser data, the setup in Prague was used. There, an electron gun is combined with a time-of-flight mass analyser. The clusters were in both cases produced via supersonic expansion. The radiosensitisers studied include 5-selenocyanato-2’-deoxyuridine (SeCNdU), nimorazole and misonidazole. All of them exhibit efficient electron capturing characteristics. SeCNdU is a potential radiosensitiser for highly proliferating cells and exhibits (SeU-yl) and CN as strongest fragment anions upon DEA, formed already at virtually 0 eV kinetic energy of the incoming electron. The formation of highly reactive species reinforce SeCNdU as a promising candidate. Nimorazole and misonidazole both exhibit an intense ion signal for the parent anion upon electron attachment. The absolute cross section at 0 eV electron energy was determined for nimorazole and is in the order of 3 1018 m2. For misonidazole, the absolute cross section is estimated to be of the same order of magnitude. The fragmentation channels upon DEA are at least one order of magnitude weaker, with NO 2 being the most intense among them. They are further quenched upon hydration of the agent. It is suggested that the radiosensitising action is caused by the associative attachment channel and the DEA products only play a minor role, opposing previous assumptions. In case of the doped neon clusters, indications for the formation of a conduction band were found in the form of an energy barrier for incoming electrons: Comparing previous results of electron attachment to pure carbon dioxide clusters with neon clusters doped with CO2 results in a blue-shift of the resonance positions by up to 0.8 eV. The effect depends on the size of the neon cluster. For molecular oxygen as the dopant, evidence was found that an incoming electron can first react with the neon cluster via an excitation event and subsequently attach to the dopant cluster. Both the study of radiosensitisers and of neon clusters should be continued. Further radiosensitisers should be studied in order to implement a model in which the effects on a physico-chemical stage are investigated and compared to radiosensitisers already in use. From such a study, potential new radiosensitisers can be derived. In the study of doped neon clustes, the solvation of different complexes with neon was investigated. Particularly, the formation of a conduction band, neon cluster - dopant interactions, and quenching of molecular processes in dopants by neon as a collision partner are analysed. The results require further investigations of additional dopants to unambiguously explain those effects

HIV-1 Replication and Pathogenesis in the Human Thymus

How HIV replicates and causes destruction of the thymus, and how to restore thymic function, are among the most important questions of HIV-1 pathogenesis and therapy in adult as well as pediatric patients. The thymus appears to function, albeit at reduced levels, throughout the life of adults, to respond to T cell depletion induced by HIV and to be suppressed by HIV. In this review, we summarize recent findings concerning HIV replication and pathogenesis in the human thymus, focusing on mechanistic insights gleaned from studies in the SCID-hu Thy/Liv mouse and human fetal-thymus organ culture (HF-TOC) models. First, we discuss HIV viral determinants and host factors involved in the replication of HIV in the thymus. Second, we consider evidence that both viral factors and host factors contribute to HIV-induced thymocyte depletion. We thus propose that multiple mechanisms, including depletion and suppression of progenitor cells, paracrine and direct lytic depletion of thymocytes, and altered thymocyte selection are involved in HIV-induced pathology in the thymus. With the SCID-hu Thy/Liv mouse and HF-TOC models, it will be important in the coming years to further clarify the virological, cell biological, and immunological mechanisms of HIV replication and pathogenesis in human thymus, and to correlate their significance in HIV disease progression

Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.

The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.This work was supported by Howard Hughes Medical Institute (https://www.hhmi.org), the Medical Research Council (https://mrc.ukri.org; MRC file reference U105188491) and a European Research Council (https://erc.europa.eu) Consolidator grant (649111) to G.S.X.E.J., and a Royal Society (https://royalsociety.org) Dorothy Hodgkin Fellowship to S.C

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file