Matrix Porewater In Crystalline Rocks: Extraction and Analysis

The chemical and isotopic characterization of porewater residing in the inter- and intragranular pore space of the low-permeability rock matrix is an important component with respect to the site characterization and safety assessment of potential host rocks for a radioactive waste disposal. The chemical and isotopic composition of porewater in such low permeability rocks has to be derived by indirect extraction techniques applied to naturally saturated rock material. In most of such indirect extraction techniques – especially in case of rocks of a porosity below about 2 vol.% – the original porewater concentrations are diluted and need to be back-calculated to in-situ concentrations. This requires a well-defined value for the connected porosity – accessible to different solutes under in-situ conditions. The derivation of such porosity values, as well as solute concentrations, is subject to various perturbations during drilling, core sampling, storage and experiments in the laboratory. The present study aims to demonstrate the feasibility of a variety of these techniques to charac-terize porewater and solute transport in crystalline rocks. The methods, which have been de-veloped during multiple porewater studies in crystalline environments, were applied on four core samples from the deep borehole DH-GAP04, drilled in the Kangerlussuaq area, Southwest Greenland, as part of the joint NWMO–Posiva–SKB Greenland Analogue Project (GAP). Potential artefacts that can influence the estimation of in situ porewater chemistry and isotopes, as well as their controls, are described in detail in this report, using specific examples from borehole DH-GAP04

The impact of testosterone, tibolone and black cohosh on purified mammary and placental 17β-hydroxysteroid dehydrogenase type 1

Abstract Context: Mammary and placental 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1). Objective: To assess the impact of testosterone, tibolone, and black cohosh on purified mammary and placental 17βHSD1. Materials and methods: 17βHSD1 was purified from human mammary gland and placenta by column chromatography, its activity was monitored by a radioactive activity assay, and the degree of purification was determined by gel electrophoresis. Photometric cofactor transformation analysis was performed to assess 17βHSD1 activity without or in presence of testosterone, tibolone and black cohosh. Results: 17βHSD1 from both sources displayed a comparable basal activity. Testosterone and tibolone metabolites inhibited purified mammary and placental 17βHSD1 activity to a different extent, whereas black cohosh had no impact. Discussion: Studies on purified enzymes reveal the individual action of drugs on local regulatory mechanisms thus helping to develop more targeted therapeutic intervention. Conclusion: Testosterone, tibolone and black cohosh display a beneficial effect on local mammary estrogen metabolism by not affecting or decreasing local estradiol exposure.

Neurotensin receptors in pancreatic ductal carcinomas.

BACKGROUND The frequent expression of neurotensin receptors (NT-R) in primaries of pancreatic ductal carcinomas has triggered the development of radioactive neurotensin analogs for possible in vivo targeting of these tumors. However, the complete lack of information regarding NT-R in liver metastases of pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) makes an in vitro study of NT-R in these tissues indispensable. METHODS Using in vitro receptor autoradiography with (125)I-[Tyr(3)]-neurotensin, NT-R were investigated in 18 primaries and 23 liver metastases of pancreatic ductal carcinomas as well as in 19 PanIN lesions. RESULTS We report here that 13 of 18 ductal carcinoma primaries and 14 of 23 liver metastases expressed NT-R. Moreover, none of the six PanIN 1B cases expressed NT-R, while two of six PanIN 2 and five of seven PanIN 3 expressed NT-R. Binding was fully displaced by the type 1 NT-R-selective antagonist SR48692, indicating that the NT-R in the tumors are of the type 1 NT-R subtype. CONCLUSIONS These in vitro data extend the currently available information on NT-R in invasive and non-invasive pancreatic ductal tumors. They suggest that type 1 NT-R may be a novel, specific marker of PanIN of higher degree. The high expression of NT-R in primaries and metastases of invasive cancer strongly support the need to develop radioactive neurotensin analogs for the diagnosis and therapy of this tumor type.

Experimental Characterization and Quantification of Cement-Bentonite Interaction using Core Infiltration Techniques Coupled with X-ray Tomography

Deep geological storage of radioactive waste foresees cementitious materials as reinforcement of tunnels and as backfill. Bentonite is proposed to enclose spent fuel canisters and as drift seals. Sand/bentonite (s/b) is foreseen as backfill material of access galleries or as drift seals. The emplacement of cementitious material next to clay material generates an enormous chemical gradient in pore-water composition that drives diffusive solute transport. Laboratory studies and reactive transport modeling predicted significant mineral alteration at and near interfaces, mainly resulting in a decrease of porosity in bentonite. The goal of this thesis was to characterize and quantify the cement/bentonite interactions both spatially and temporally in laboratory experiments. A newly developed mobile X-ray transparent core infiltration device was used to perform X-ray computed tomography (CT) scans without interruption of running experiments. CT scans allowed tracking the evolution of the reaction plume and changes in core volume/diameter/density during the experiments. In total 4 core infiltration experiments were carried out for this study with the compacted and saturated cores consisting of MX-80 bentonite and sand/MX-80 bentonite mixture (s/b; 65/35%). Two different high-pH cementitious pore-fluids were infiltrated: a young (early) ordinary Portland cement pore-fluid (APWOPC; K+–Na+–OH-; pH 13.4; ionic strength 0.28 mol/kg) and a young ‘low-pH’ ESDRED shotcrete pore-fluid (APWESDRED; Ca2+–Na+–K+–formate; pH 11.4; ionic strength 0.11 mol/kg). The experiments lasted between 1 and 2 years. In both bentonite experiments, the hydraulic conductivity was strongly reduced after switching to high-pH fluids, changing eventually from an advective to a diffusion-dominated transport regime. The reduction was mainly induced by mineral precipitation and possibly partly also by high ionic strength pore-fluids. Both bentonite cores showed a volume reduction and a resulting transient flow in which pore-water was squeezed out during high-pH infiltration. The outflow chemistry was characterized by a high ionic strength, while chloride in the initial pore water got replaced as main anionic charge carrier by sulfate, originating from gypsum dissolution. The chemistry of the high-pH fluids got strongly buffered by the bentonite, consuming hydroxide and in case of APWESDRED also formate. Hydroxide got consumed by mineral reactions (saponite and possibly talc and brucite precipitation), while formate being affected by bacterial degradation. Post-mortem analysis showed reaction zones near the inlet of the bentonite core, characterized by calcium and magnesium enrichment, consisting predominately of calcite and saponite, respectively. Silica got enriched in the outflow, indicating dissolution of silicate-minerals, identified as preferentially cristobalite. In s/b, infiltration of APWOPC reduced the hydraulic conductivity strongly, while APWESDRED infiltration had no effect. The reduction was mainly induced by mineral precipitation and probably partly also by high ionic strength pore-fluids. Not clear is why the observed mineral precipitates in the APWESDRED experiment had no effect on the fluid flow. Both s/b cores showed a volume expansion along with decreasing ionic strengths of the outflow, due to mineral reactions or in case of APWESDRED infiltration also mediated by microbiological activity, consuming hydroxide and formate, respectively. The chemistry of the high-pH fluids got strongly buffered by the s/b. In the case of APWESDRED infiltration, formate reached the outflow only for a short time, followed by enrichment in acetate, indicating most likely biological activity. This was in agreement to post-mortem analysis of the core, observing black spots on the inflow surface, while the sample had a rotten-egg smell indicative of some sulfate reduction. Post-mortem analysis showed further in both cores a Ca-enrichment in the first 10 mm of the core due to calcite precipitation. Mg-enrichment was only observed in the APWOPC experiment, originating from newly formed saponite. Silica got enriched in the outflow of both experiments, indicating dissolution of silicate-minerals, identified in the OPC experiment as cristobalite. The experiments attested an effective buffering capacity for bentonite and s/b, a progressing coupled hydraulic-chemical sealing process and also the preservation of the physical integrity of the interface region in this setup with a total pressure boundary condition on the core sample. No complete pore-clogging was observed but the hydraulic conductivity got rather strongly reduced in 3 experiments, explained by clogging of the intergranular porosity (macroporosity). Such a drop in hydraulic conductivity may impact the saturation time of the buffer in a nuclear waste repository, although the processes and geometry will be more complex in repository situation.

Interaction of corroding iron with bentonite in the ABM1 experiment at Äspö, Sweden: A microscopic approach

Bentonite and iron metals are common materials proposed for use in deep-seated geological repositories for radioactive waste. The inevitable corrosion of iron leads to interaction processes with the clay which may affect the sealing properties of the bentonite backfill. The objective of the present study was to improve our understanding of this process by studying the interface between iron and compacted bentonite in a geological repository-type setting. Samples of MX-80 bentonite samples which had been exposed to an iron source and elevated temperatures (up to 115ºC) for 2.5 y in an in situ experiment (termed ABM1) at the Äspö Hard Rock Laboratory, Sweden, were investigated by microscopic means, including scanning electron microscopy, μ-Raman spectroscopy, spatially resolved X-ray diffraction, and X-ray fluorescence. The corrosion process led to the formation of a ~100 mm thick corrosion layer containing siderite, magnetite, some goethite, and lepidocrocite mixed with the montmorillonitic clay. Most of the corroded Fe occurred within a 10 mm-thick clay layer adjacent to the corrosion layer. An average corrosion depth of the steel of 22–35 μm and an average Fe2+ diffusivity of 1–26×10–13 m2/s were estimated based on the properties of the Fe-enriched clay layer. In that layer, the corrosion-derived Fe occurred predominantly in the clay matrix. The nature of this Fe could not be identified. No indications of clay transformation or newly formed clay phases were found. A slight enrichment of Mg close to the Fe–clay contact was observed. The formation of anhydrite and gypsum, and the dissolution of some SiO2 resulting from the temperature gradient in the in situ test, were also identified. © 2014, Clay Minerals Society. All right reserved.

Quantification of water content across a cement-clay interface using high resolution neutron radiography

In many designs for radioactive waste repositories, cement and clay will come into direct contact. The geochemical contrast between cement and clay will lead to mass fluxes across the interface, which consequently results in alteration of structural and transport properties of both materials that may affect the performance of the multi-barrier system. We present an experimental approach to study cement-clay interactions with a cell to accommodate small samples of cement and clay. The cell design allows both in situ measurement of water content across the sample using neutron radiography and measurement of transport parameters using through-diffusion tracer experiments. The aim of the high- resolution neutron radiography experiments was to monitor changes in water content (porosity) and their spatial extent. Neutron radiographs of several evolving cement-clay interfaces delivered quantitative data which allow resolving local water contents within the sample domain. In the present work we explored the uncertainties of the derived water contents with regard to various input parameters and with regard to the applied image correction procedures. Temporal variation of measurement conditions created absolute uncertainty of the water content in the order of ±0.1 (m3/m3), which could not be fully accounted for by correction procedures. Smaller relative changes in water content between two images can be derived by specific calibrations to two sample regions with different, invariant water contents.

Flux control of sulphate assimilation in Arabidopsis thaliana : adenosine 5′-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied l-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5′-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing l-cysteine to the nutrient solution increased internal cysteine, γ-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm l-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm l-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of l-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm l-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using 35SO42– in the presence of 0.5 mm l-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.

Particle Accelerators and Detectors for medical Diagnostics and Therapy

This Habilitationsschrift (Habilitation thesis) is focused on my research activities on medical applications of particle physics and was written in 2013 to obtain the Venia Docendi (Habilitation) in experimental physics at the University of Bern. It is based on selected publications, which represented at that time my major scientific contributions as an experimental physicist to the field of particle accelerators and detectors applied to medical diagnostics and therapy. The thesis is structured in two parts. In Part I, Chapter 1 presents an introduction to accelerators and detectors applied to medicine, with particular focus on cancer hadrontherapy and on the production of radioactive isotopes. In Chapter 2, my publications on medical particle accelerators are introduced and put into their perspective. In particular, high frequency linear accelerators for hadrontherapy are discussed together with the new Bern cyclotron laboratory. Chapter 3 is dedicated to particle detectors with particular emphasis on three instruments I contributed to propose and develop: segmented ionization chambers for hadrontherapy, a proton radiography apparatus with nuclear emulsion films, and a beam monitor detector for ion beams based on doped silica fibres. Selected research and review papers are contained in Part II. For copyright reasons, they are only listed and not reprinted in this on-line version. They are available on the websites of the journals.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Effect of mannitol and cold treatments on phosphate uptake and protein phosphorylation in Lemna minor (L.) plants

This report is aimed at elucidating the effect of mannitol and cold treatments on P uptake and protein phosphorylation in Lemna minor plants. Duckweed p lants were incu bated in the presence of [32P]or [33P]Pi in half-strength phosphate deprived E-medium under constant light regime for 1.5 h. Total plant protein extracts (pellet and supernatant) were then prepared and subjected to IEF x SDS-PAGE. To analyse the effect of the stresses on P uptake and protein labelling, Lemna minor plants were preincubated with 0.1, 0.5 mol · L-1 mannitol and at 4°C respectively, for 4 hours, before adding labelled orthophosphate. The results show that the general protein phosphorylation (including LHCII) is related to the level of P uptake. Radioactive phosphate incorporation is stimulated by a low concentration of mannitol (0.1 mol · L-1) but reduced by 0.5 mol · L-1 mannitol and cold stress in planta. The labelling into proteins is affected neither when stresses were applied to the plants after incubation with labelled orthophosphate, nor after in vitro protein phosphorylation. This indicates that general protein kinase activities in vivo are strictly limited by P uptake. A marked accumulation of soluble hexoses (mainly sucrose, glucose, and fructose) is observed under imposed stress, suggesting that the inhibition of P uptake in response to hyperosmotic and cold stresses is mediated by sugar accumulation in situ. However, metabolisable sugars like glucose did not alter the entry of phosphate at concentrations of 0.5 mol · L-1, showing that the chemical nature of the osmoticum influences P uptake.

Sulfate assimilation in higher plants. Characterization of a stable intermediate in the adenosine 5′-phosphosulfate reductase reaction

The enzyme catalysing the reduction of adenosine 5′-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 °C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2–3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5′-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 °C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2–3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2–3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.

Cosmogenic activation of xenon and copper

Rare event search experiments using liquid xenon as target and detection medium require ultra-low background levels to fully exploit their physics potential. Cosmic ray induced activation of the detector components and, even more importantly, of the xenon itself during production, transportation and storage at the Earth's surface, might result in the production of radioactive isotopes with long half-lives, with a possible impact on the expected background. We present the first dedicated study on the cosmogenic activation of xenon after 345 days of exposure to cosmic rays at the Jungfraujoch research station at 3470m above sea level, complemented by a study of copper which has been activated simultaneously. We have directly observed the production of 7Be, 101Rh, 125Sb, 126I and 127Xe in xenon, out of which only 125Sb could potentially lead to background for a multi-ton scale dark matter search. The production rates for five out of eight studied radioactive isotopes in copper are in agreement with the only existing dedicated activation measurement, while we observe lower rates for the remaining ones. The specific saturation activities for both samples are also compared to predictions obtained with commonly used software packages, where we observe some underpredictions, especially for xenon activation.