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In the absence of sources of rapidly varying field, the study of the potential distribution determined, in a linear medium, by a geometrically complex grounding may be conducted by applying the method of superposition of the effects, known as method of subareas of Maxwell. Such approach can be usefully employed in an educational tool intended for students of electrical engineering.

This paper presents a real-time monitoring system for PV power plants based on a microcontroller Atmega328P-PU. The proposed system, whose performances can be relatively close to those achieved with commercial monitoring systems, is simple, low-cost and open source technology-based. In addition, a user interface, capable to communicate through a wireless network by using a standard communication protocol IEEE 802.15.1, is here presented. From the experimental tests described and discussed in this paper it is demonstrated that the proposed monitoring system provides, with adequate accuracy, all the information about the operating status of the PV plant, even during fault conditions.

In this paper a wind electrical power generation system for Distributed on Site (DoS) applications is proposed. This system was developed and conceived in order to guarantee simple installation, use and service, obtaining a product that can be easily industrialized and put into the market with limited costs. The field of application of such an electrical generation system is addressed towards domestic or at the most toward condominium applications concerning moreover the opportunity of working in addition to combined photovoltaic and solar-thermal systems to enhance the renewable energy generation at house level (DoS). The proposed system has its hot spot in the blades shape, in the wind flux canalization system and in the permanent magnet synchronous generator (PMSG) optimized for this specific purpose. In fact with the proposed system intends to search the best aerodynamic profile and electrical generator to ensure the slightest encumbrance and the lowest weight. For this purpose the traditional and classical tangential wind turbine design, equipped with large blades, is overturned in favour of the use of numerous little blades. A further peculiarity of the system is the expected increase of its efficiency thanks to the presence of the wind flux channelling system with the elimination of the part opposing the wind and thanks to the adoption of a specifically designed PMSG.

This paper shows the performance of a PV system under shadow projection on PV panels. The reconfiguration techniques, employing a microprocessor-based system, modifies the layout of the PV plant by physically changing the connections among modules, in order to recover the loss of power due to the shadows. Different cases have been considered to evaluate the increase of power.

In this paper the behavior of a tower and its grounding system, subjected to a lightning, is faced. Finite difference time domain (FDTD) method has been chosen in order to study the non-linear time domain behavior of the system. The electromagnetic problem has been described by using two type of first order time derivative equations: Maxwell's equations and Telegraph equations. Aim of this work is to evaluate the possibility of a flashover between tower and power line, by considering different cases of study.

International audience; Proton and ion beams are radiotherapy modalities of increasing importance and interest. Because of the different biological dose response of these radiations as compared with high-energy photon beams, the current approach of treatment prescription is based on the product of the absorbed dose to water and a biological weighting factor, but this is found to be insufficient for providing a generic method to quantify the biological outcome of radiation. It is therefore suggested to define new dosimetric quantities that allow a transparent separation of the physical processes from the biological ones. Given the complexity of the initiation and occurrence of biological processes on various time and length scales, and given that neither microdosimetry nor nanodosimetry on their own can fully describe the biological effects as a function of the distribution of energy deposition or ionization, a multiscale approach is needed to lay the foundation for the aforementioned new physical quantities relating track structure to relative biological effectiveness in proton and ion beam therapy. This article reviews the state-of-the-art microdosimetry, nanodosimetry, track structure simulations, quantification of reactive species, reference radiobiological data, cross-section data and multiscale models of biological response in the context of realizing the new quantities. It also introduces the European metrology project, Biologically Weighted Quantities in Radiotherapy, which aims to investigate the feasibility of establishing a multiscale model as the basis of the new quantities. A tentative generic expression of how the weighting of physical quantities at different length scales could be carried out is presented. © 2015 The Authors.

Ionising radiation has been an important part of cancer treatment for almost a century, being used in external-beam radiotherapy, brachytherapy, and targeted radionuclide therapy. At the molecular and cellular level, cell killing has been attributed to deposition of energy from the radiation in the DNA within the nucleus, with production of DNA double-strand breaks playing a central part. However, this DNA-centric model has been questioned because cell-death pathways, in which direct relations between cell killing and DNA damage diverge, have been reported. These pathways include membrane-dependent signalling pathways and bystander responses (when cells respond not to direct radiation exposure but to the irradiation of their neighbouring cells). New insights into mechanisms of these responses coupled with technological advances in targeting of cells in experimental systems with microbeams have led to a reassessment of the model of how cells are killed by ionising radiation. This review provides an update on these mechanisms.

This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells.

Radiation microbeams represent a unique and powerful tool to study and quantify the effects of precise doses of radiation delivered with micron precision to selected biological samples. The Gray Cancer Institute has developed two independent but complementary micro-irradiation facilities, specifically developed for the targeted irradiation of cells and structured tissues; a charged-particle microbeam that uses collimated protons or helium ions and an ultra-soft X-ray microprobe. The ultra-soft X-ray facility employs a focused electron bombardments source to produce a near monochromatic C K X-ray beam. Highly efficient zone plates optimised for the appropriate wavelength are used to focus the characteristic X-rays into a sub-micron spot. The facility is also equipped with a three-axis micro-positioning stage, an epi-fluorescent UV microscope with intensified CCD camera coupled to a fast PC for a automatic, fast and accurate samples recognition and alignment with the probe. Recent experiments have been directed to investigate the bystander effect by irradiating only one cell within a population of V79 cells that are subsequently individually revisited for colony formation. A clear bystander effect has been detected (- 10 % reduction in survival) when a single cell has been irradiated. The effect is triggered by very low doses (∼ 100) mGy) and it is largely dose independent.

Purpose The biological optimization of proton therapy can be achieved only through a detailed evaluation of relative biological effectiveness (RBE) variations along the full range of the Bragg curve. The clinically used RBE value of 1.1 represents a broad average, which disregards the steep rise of linear energy transfer (LET) at the distal end of the spread-out Bragg peak (SOBP). With particular attention to the key endpoint of cell survival, our work presents a comparative investigation of cell killing RBE variations along monoenergetic (pristine) and modulated (SOBP) beams using human normal and radioresistant cells with the aim to investigate the RBE dependence on LET and intrinsic radiosensitvity. Methods and Materials Human fibroblasts (AG01522) and glioma (U87) cells were irradiated at 6 depth positions along pristine and modulated 62-MeV proton beams at the INFN-LNS (Catania, Italy). Cell killing RBE variations were measured using standard clonogenic assays and were further validated using Monte Carlo simulations and the local effect model (LEM). Results We observed significant cell killing RBE variations along the proton beam path, particularly in the distal region showing strong dose dependence. Experimental RBE values were in excellent agreement with the LEM predicted values, indicating dose-averaged LET as a suitable predictor of proton biological effectiveness. Data were also used to validate a parameterized RBE model. Conclusions The predicted biological dose delivered to a tumor region, based on the variable RBE inferred from the data, varies significantly with respect to the clinically used constant RBE of 1.1. The significant RBE increase at the distal end suggests also a potential to enhance optimization of treatment modalities such as LET painting of hypoxic tumors. The study highlights the limitation of adoption of a constant RBE for proton therapy and suggests approaches for fast implementation of RBE models in treatment planning.

This paper shows the economic convenience of the application of a reconfiguration system for photovoltaic plants, considering the incentives system in Italy and its recent modifications. The reconfiguration is an alternative to the Distributed Maximum Power Point Tracking, DMPPT, technique used to overcome the mismatch problem in Photovoltaic, PV, plants. The reconfiguration techniques employ a microprocessor-based system that modifies the layout of the PV plant by physically changing the connections among modules. After the explanation of the working principle of the PV reconfiguration system allowing the improvement of the efficiency of the same plant and the relevant installation, the main strength and weakness points of the investment are evidenced in relation to different incentives policies.

ELI-Beamlines is one of the pillars of the pan-European project ELI (Extreme Light Infrastructure). It will be an ultra high-intensity, high repetition-rate, femtosecond laser facility whose main goal is generation and applications of high-brightness X-ray sources and accelerated charged particles in different fields. Particular care will be devoted to the potential applicability of laser-driven ion beams for medical treatments of tumors. Indeed, such kind of beams show very interesting peculiarities and, moreover, laser-driven based accelerators can really represent a competitive alternative to conventional machines since they are expected to be more compact in size and less expensive. The ELIMED project was launched thanks to a collaboration established between FZU-ASCR (ELI-Beamlines) and INFN-LNS researchers. Several European institutes have already shown a great interest in the project aiming to explore the possibility to use laser-driven ion (mostly proton) beams for several applications with a particular regard for medical ones. To reach the project goal several tasks need to be fulfilled, starting from the optimization of laser-target interaction to dosimetric studies at the irradiation point at the end of a proper designed transport beam-line. Researchers from LNS have already developed and successfully tested a high-dispersive power Thomson Parabola Spectrometer, which is the first prototype of a more performing device to be used within the ELIMED project. Also a Magnetic Selection System able to produce a small pencil beam out of a wide energy distribution of ions produced in laser-target interaction has been realized and some preliminary work for its testing and characterization is in progress. In this contribution the status of the project will be reported together with a short description of the of the features of device recently developed. Conference on Plasma Physics by Laser and Applications (PPLA), Oct 02-04, 2013, Univ Salento, Lecce, Italy

Cellular micro-irradiation is now recognised as a powerful technique for understanding how ionising radiation interacts with living cells and tissues. Charged-particle microbeams are uniquely capable of delivering single, or counted multiple particles to selected sub-cellular targets. This capability is particularly useful for studying the risks associated with environmental exposures to α-particle emitting isotopes (such as radon) where exposed cells within the body are unlikely to receive more than one particle traversal. Microbeam methods are also seen as highly appropriate for studying the so-called `bystander effect' (where unirradiated cells respond to signals transmitted by irradiated neighbours). Using the Gray Laboratory microbeam, we have been able to demonstrate a significant increase in the levels of cell death and DNA damage in a population of cells after irradiating just a few cells within a population. Also, by targeting the cell cytoplasm, we have shown that intra-cellular signalling between the cytoplasm and nucleus can cause DNA damage, showing that direct DNA damage is not required to observe radiation induced effect in cells.

Understanding the effects of single-particles from conventional radiation biology experiments is problematic due to the stochastics of particle tracks. This complicates the determinations of risk associated with low doses. We have developed a charged particle microbeam, which allows individually counted particles to be delivered to precise cellular locations. The system is capable of delivering a single charged particle with99% efficiency. Of these particles 90% are delivered with a resolution of +/- 2 micrometers and 96% with a resolution of +/- 5 micrometers. We have carried out preliminary studies in Chinese hamster V79 cells to monitor the effectiveness of low energy protons at inducing cytological damage. We have used the micronucleus assay as a measure of predominantly lethal chromosome damage. The effects of a single 3.2 MeV proton delivered individually to cells could be measured, with less than 2% of the exposed cells producing micronuclei 24 hours later. The yield of micronuclei formation was essentially linear up to the highest dose (30 particles per cell nucleus) delivered. Ultimately, the ability to target particles to different parts of the cell nucleus may start to impact on models available for chromosome aberration formation and chromosomal Organisation and mechanisms underlying genomic instability.

We are developing two independent, but complementary microbeams for irradiating cells individually in vitro. Firstly, a charged-particle microbeam that uses a fine-bore glass capillary, combined with a transmission detector to precisely irradiate cells with exact numbers of energetic charge-particles and secondly, a soft X-ray microprobe that produces a very fine beam of carbon-K (278 eV) ultrasoft X-rays, focused to a spot size

Summary Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage., Graphical Abstract, Highlights • BRCA1 forms a complex with mRNA splicing proteins following DNA damage • The BRCA1-mRNA splicing complex promotes the stability of genes involved in the DDR • BRCA1-dependent splicing of DDR genes maintains their protein expression • The BRCA1-mRNA splicing complex is required for DNA repair and genomic stability, BRCA1 functions to maintain genomic integrity by forming multiple protein complexes with different functions. Here, Savage et al. report the identification of a BRCA1 protein complex that promotes the mRNA splicing and expression of genes required for maintaining genomic stability.

Studies regarding the radiobiological effects of low dose radiation, microbeam irradiation services have been developed in the world and today laser acceleration of protons and heavy ions may be used in radiation therapy. The application of different facilities is essential for studying bystander effects and relating signalling phenomena in different cells or tissues. In particular the use of ion beams results advantageous in cancer radiotherapy compared to more commonly used X-rays, since the ability of ions in delivering lethal amount of doses into the target tumour avoiding or limiting damage to the contiguous healthy tissues. At the INFN-LNS in Catania, a multidisciplinary radiobiology group is strategically structured aimed to develop radiobiological research, finalised to therapeutic applications, compatible with the use of high dose laser-driven ion beams. The characteristic non-continuous dose rates with several orders of magnitude of laser-driven ion beams makes this facility very interesting in the cellular systems' response to ultra-high dose rates with non-conventional pulse time intervals cellular studies. Our group have projected to examine the effect of high dose laser-driven ion beams on two cellular types: foetal fibroblasts (normal control cells) and DU145 (prostate cancer cells), studying the modulation of some different bio-molecular parameters, in particular cell proliferation and viability, DNA damage, redox cellular status, morphological alterations of both the cytoskeleton components and some cell organelles and the possible presence of apoptotic or necrotic cell death. Our group performed preliminary experiments with high energy (60 MeV), dose rate of 10 Gy/min, doses of 1, 2, 3 Gy and LET 1 keV/mu m on human foetal fibroblasts (control cells). We observed that cell viability was not influenced by the characteristics of the beam, the irradiation conditions or the analysis time. Conversely, DNA damage was present at time 0, immediately following irradiation in a dose-dependent manner. The analysis of repair capability showed that the cells irradiated with 1 and 2 Gy almost completely recovered from the damage, but not, however, 3 Gy treated cells in which DNA damage was not recovered. In addition, the results indicate the importance of the use of an appropriate control in radiobiological in vitro analysis.

The potential that laser based particle accelerators offer to solve sizing and cost issues arising with conventional proton therapy has generated great interest in the understanding and development of laser ion acceleration, and in investigating the radiobiological effects induced by laser accelerated ions. Laser-driven ions are produced in bursts of ultra-short duration resulting in ultra-high dose rates, and an investigation at Queen's University Belfast was carried out to investigate this virtually unexplored regime of cell rdaiobiology. This employed the TARANIS terawatt laser producing protons in the MeV range for proton irradiation, with dose rates exceeding 109 Gys-1 on a single exposure. A clonogenic assay was implemented to analyse the biological effect of proton irradiation on V79 cells, which, when compared to data obtained with the same cell line irradiated with conventionally accelerated protons, was found to show no significant difference. A Relative Biological effectiveness of 1.4±0.2 at 10 % Survival Fraction was estimated from a comparison with a 225 kVp X-ray source. © 2013 SPIE.

Biological validation of new radiotherapy modalities is essential to understand their therapeutic potential. Antiprotons have been proposed for cancer therapy due to enhanced dose deposition provided by antiproton-nucleon annihilation. We assessed cellular DNA damage and relative biological effectiveness (RBE) of a clinically relevant antiproton beam. Despite a modest LET (~19 keV/μm), antiproton spread out Bragg peak (SOBP) irradiation caused significant residual γ-H2AX foci compared to X-ray, proton and antiproton plateau irradiation. RBE of ~1.48 in the SOBP and ~1 in the plateau were measured and used for a qualitative effective dose curve comparison with proton and carbon-ions. Foci in the antiproton SOBP were larger and more structured compared to X-rays, protons and carbon-ions. This is likely due to overlapping particle tracks near the annihilation vertex, creating spatially correlated DNA lesions. No biological effects were observed at 28–42 mm away from the primary beam suggesting minimal risk from long-range secondary particles.

CORRIGENDUM: Biological consequences of nanoscale energy deposition near irradiated heavy atom nanoparticles

SIGNIFICANCE:Ionizing radiation (IR) can induce a wide range of unique deoxyribonucleic acid (DNA) lesions due to the spatiotemporal correlation of the ionization produced. Of these, DNA double strand breaks (DSBs) play a key role. Complex mechanisms and sophisticated pathways are available within cells to restore the integrity and sequence of the damaged DNA molecules.RECENT ADVANCES:Here we review the main aspects of the DNA DSB repair mechanisms with emphasis on the molecular pathways, radiation-induced lesions, and their significance for cellular processes.CRITICAL ISSUES:Although the main characteristics and proteins involved in the two DNA DSB repair processes present in eukaryotic cells (homologous recombination and nonhomologous end-joining) are reasonably well established, there are still uncertainties regarding the primary sensing event and their dependency on the complexity, location, and time of the damage. Interactions and overlaps between the different pathways play a critical role in defining the repair efficiency and determining the cellular functional behavior due to unrepaired/miss-repaired DNA lesions. The repair pathways involved in repairing lesions induced by soluble factors released from directly irradiated cells may also differ from the established response mechanisms.FUTURE DIRECTIONS:An improved understanding of the molecular pathways involved in sensing and repairing damaged DNA molecules and the role of DSBs is crucial for the development of novel classes of drugs to treat human diseases and to exploit characteristics of IR and alterations in tumor cells for successful radiotherapy applications.

The ultra short duration of laser-driven multi-MeV ion bursts offers the possibility of radiobiological studies at extremely high dose rates. Employing the TARANIS Terawatt laser at Queen's University, the effect of proton irradiation at MeV-range energies on live cells has been investigated at dose rates exceeding 10 Gy/s as a single exposure. A clonogenic assay showed consistent lethal effects on V-79 live, cells, which, even at these dose rates, appear to be in line with previously published results employing conventional sources. A Relative Biological Effectiveness (RBE) of 1.4±0.2 at 10% survival is estimated from a comparison with a 225 kVp X-ray source. © 2013 AIP Publishing LLC.

It is now widely accepted that intercellular communication can cause significant variations in cellular responses to genotoxic stress. The radiation-induced bystander effect is a prime example of this effect, where cells shielded from radiation exposure see a significant reduction in survival when cultured with irradiated cells. However, there is a lack of robust, quantitative models of this effect which are widely applicable. In this work, we present a novel mathematical model of radiation-induced intercellular signalling which incorporates signal production and response kinetics together with the effects of direct irradiation, and test it against published data sets, including modulated field exposures. This model suggests that these so-called "bystander" effects play a significant role in determining cellular survival, even in directly irradiated populations, meaning that the inclusion of intercellular communication may be essential to produce robust models of radio-biological outcomes in clinically relevant in vivo situations.

For the delivery of intensity-modulated radiation therapy (IMRT), highly modulated fields are used to achieve dose conformity across a target tumour volume. Recent in vitro evidence has demonstrated significant alterations in cell survival occurring out-of-field which cannot be accounted for on the basis of scattered dose. The radiobiological effect of area, dose and dose-rate on out-of-field cell survival responses following exposure to intensity-modulated radiation fields is presented in this study. Cell survival was determined by clonogenic assay in human prostate cancer (DU-145) and primary fibroblast (AG0-1522) cells following exposure to different modulated field configurations delivered using a X-Rad 225 kVp x-ray source. Uniform survival responses were compared to in- and out-of-field responses in which 25-99% of the cell population was shielded. Dose delivered to the out-of-field region was varied from 1.6-37.2% of that delivered to the in-field region using different levels of brass shielding. Dose rate effects were determined for 0.2-4 Gy min⁻¹ for uniform and modulated exposures with no effect seen in- or out-of-field. Survival responses showed little dependence on dose rate and area in- and out-of-field with a trend towards increased survival with decreased in-field area. Out-of-field survival responses were shown to scale in proportion to dose delivered to the in-field region and also local dose delivered out-of-field. Mathematical modelling of these findings has shown survival response to be highly dependent on dose delivered in- and out-of-field but not on area or dose rate. These data provide further insight into the radiobiological parameters impacting on cell survival following exposure to modulated irradiation fields highlighting the need for refinement of existing radiobiological models to incorporate non-targeted effects and modulated dose distributions.

The ultrashort duration of laser-driven multi-MeV ion bursts offers the possibility of radiobiological studies at extremely high dose rates. Employing the TARANIS Terawatt laser at Queen's University, the effect of proton irradiation at MeV-range energies on live cells has been investigated at dose rates exceeding 109Gy/s as a single exposure. A clonogenic assay showed consistent lethal effects on V-79 live cells, which, even at these dose rates, appear to be in line with previously published results employing conventional sources. A Relative Biological Effectiveness (RBE) of 1.4±0.2 at 10% survival is estimated from a comparison with a 225 kVp X-ray source.

Background and purpose The addition of gold nanoparticles (GNPs) to tumours leads to an increase in dose due to their high density and energy absorption coefficient, making it a potential radiosensitiser. However, experiments have observed radiosensitisations significantly larger than the increase in dose alone, including at megavoltage energies where gold's relative energy absorption is lowest. This work investigates whether GNPs create dose inhomogeneities on a sub-cellular scale which combine with non-linear dose dependence of cell survival to be the source of radiosensitisation at megavoltage energies. Materials and methods Monte Carlo simulations were carried out to calculate dose in the vicinity of a single GNP on the nanoscale. The effect of this nanoscale dose distribution was then modelled for MDA-MB-231 cells exposed to 2nm GNPs, and compared to experimental results. Results Dramatic dose inhomogeneities occur around GNPs exposed to megavoltage radiation. When analysed using the Local Effect Model, these inhomogeneities lead to significant radiosensitisation, in agreement with experimental results. Conclusions This work suggests that GNP radiosensitisation is driven by inhomogeneities in dose on the nanoscale, rather than changes in dose over the entire cell, which may contribute to the similar radiosensitisation observed in megavoltage and kilovoltage experiments. The short range of these inhomogeneities and the variation in enhancement in different cells suggests sub-cellular localisation is important in determining GNP radiosensitisation.

In view of their properties, laser-driven ion beams have the potential to be employed in innovative applications in the scientific, technological and medical areas. Among these, a particularly high-profile application is particle therapy for cancer treatment, which however requires significant improvements from current performances of laser-driven accelerators. The focus of current research in this field is on developing suitable strategies enabling laser-accelerated ions to match these requirements, while exploiting some of the unique features of a laser-driven process. LIBRA is a UK-wide consortium, aiming to address these issues, and develop laser-driven ion sources suitable for applicative purposes, with a particular focus on biomedical applications. We will report on the activities of the consortium aimed to optimizing the properties of the beams, by developing and employing advanced targetry and by exploring novel acceleration regimes enabling production of beams with reduced energy spread. Employing the TARANIS Terawatt laser at Queen's University, we have initiated a campaign investigating the effects of proton irradiation of biological samples at extreme dose rates (> 109 Gy/s).

Non-targeted effects induced by ionizing radiation have been demonstrated both in vitro and in vivo. Previously, we have also demonstrated the existence of non-targeted effects in intact Arabidopsis seeds following low-energy heavy-ion radiation. In the present study, 6.5 MeV protons with 8 × 10(11) ions/cm(2) and 2 × 10(11) ions/cm(2) fluence respectively were used to irradiate non-shielded or partial-shielded Arabidopsis seeds to further explore the mechanisms which regulate in vivo non-targeted effects and to investigate the difference between damage caused by non-targeted effects and direct irradiation. Results showed that excess reactive oxygen species (ROS) are present in the non-irradiated part of the partially irradiated samples, indicating that in vivo non-targeted effects can promote the generation of excess metabolic ROS in the non-irradiated shoot apical meristem/root apical meristem cells. Furthermore, pretreatment with 0.5% ROS scavenger dimethyl sulfoxide (DMSO) or 0.02 mM reactive nitrogen species (RNS) scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) significantly suppresses the non-targeted effects in the partially irradiated samples, while in the whole-body irradiated samples, the cPTIO pretreatment has no effect. On the other hand using antioxidant enzyme assays, superoxide dismutase activity was found to increase for partial irradiated samples and decrease for the whole-body exposed seeds. Taken together, these results implicate that damage caused by non-targeted effects is different from that induced by direct irradiation in vivo. Metabolic products such as ROS and RNS are involved in the in vivo non-targeted effects.

Gold nanoparticles (GNPs) are being proposed as contrast agents to enhance X-ray imaging and radiotherapy, seeking to take advantage of the increased X-ray absorption of gold compared to soft tissue. However, there is a great discrepancy between physically predicted increases in X-ray energy deposition and experimentally observed increases in cell killing. In this work, we present the first calculations which take into account the structure of energy deposition in the nanoscale vicinity of GNPs and relate this to biological outcomes, and show for the first time good agreement with experimentally observed cell killing by the combination of X-rays and GNPs. These results are not only relevant to radiotherapy, but also have implications for applications of heavy atom nanoparticles in biological settings or where human exposure is possible because the localised energy deposition high-lighted by these results may cause complex DNA damage, leading to mutation and carcinogenesis.

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organisation is still unclear despite its decisive role in determining the fate of the damaged cell. Revealing the dynamic sequence of the repair proteins is therefore critical in understanding how the DNA repair mechanisms work. There are also still open questions regarding the possible movement of damaged chromatin domains and its role as trigger for lesion recognition and signalling in the DNA repair context. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. We have followed the development of radiation-induced foci for three DNA damage markers (i.e. γ-H2AX, 53BP1 and hSSB1) using normal fibroblasts (AG01522), human breast adenocarcinoma cells (MCF7) and human fibrosarcoma cells (HT1080) stably transfected with yellow fluorescent protein fusion proteins following irradiation with the QUB X-ray microbeam (carbon X-rays

Radiotherapy employs ionizing radiation to induce lethal DNA lesions in cancer cells while minimizing damage to healthy tissues. Due to their pattern of energy deposition, better therapeutic outcomes can, in theory, be achieved with ions compared to photons. Antiprotons have been proposed to offer a further enhancement due to their annihilation at the end of the path. The work presented here aimed to establish and validate an experimental procedure for the quantification of plasmid and genomic DNA damage resulting from antiproton exposure. Immunocytochemistry was used to assess DNA damage in directly and indirectly exposed human fibroblasts irradiated in both plateau and Bragg peak regions of a 126 MeV antiproton beam at CERN. Cells were stained post irradiation with an anti-γ-H2AX antibody. Quantification of the γ-H2AX foci-dose relationship is consistent with a linear increase in the Bragg peak region. A qualitative analysis of the foci detected in the Bragg peak and plateau region indicates significant differences highlighting the different severity of DNA lesions produced along the particle path. Irradiation of desalted plasmid DNA with 5 Gy antiprotons at the Bragg peak resulted in a significant portion of linear plasmid in the resultant solution.

Microbeams have undergone a renaissance since their introduction and early use in the mid 60s. Recent advances in imaging, software and beam delivery have allowed rapid technological developments in microbeams for use in a range of experimental studies. The resurgence in the use of microbeams since the mid 90s has coincided with major changes in our understanding of how radiation interacts with cells. In particular, the evidence that bystander responses occur, where cells not directly irradiated can respond to irradiated neighbours, has brought about the evolution of new models of radiation response. Although these processes have been studied using a range of experimental approaches, microbeams offer a unique route by which bystander responses can be elucidated. Without exception, all of the microbeams currently active internationally have studied bystander responses in a range of cell and tissue models. Together these studies have considerably advanced our knowledge of bystander responses and the underpinning mechanisms. Much of this has come from charged particle microbeam studies, but increasingly, X-ray and electron microbeams are starting to contribute quantitative and mechanistic information on bystander effects. A recent development has been the move from studies with 2-D cell culture models to more complex 3-D systems where the possibilities of utilizing the unique characteristics of microbeams in terms of their spatial and temporal delivery will make a major impact.

Cells have evolved complex processes to maintain the stability of their genomes. In response to genotoxic stress, the DNA damage response (DDR) is activated whereby a series of interlinked sensor processes signal to a panel of repair pathways, which can attempt to repair the damage. Recent studies have shown compelling evidence for the activation of DDR by ionising radiation even when radiation is not directly deposited in the DNA within the nucleus via bystander responses. Several groups have reported activation of a DDR response in bystander cells involving γ-H2AX formation and the potential formation of DNA doublestrand breaks leading to mutations, chromosomal aberrations, and cell death. Earlier studies by Little and colleagues have proposed that clustering of damage including base damage at DNA replication forks may be important in bystander cells. In further studies, we now have evidence in bystander cells that the initial phosphorylation of H2AX is performed by the ataxia-telangiectasia and Rad3-related protein (ATR) rather that ataxia-telangiectasia mutated protein (ATM) or DNA-dependent protein kinase (DNA-PK). This action occurs predominantly in S-phase cells and supports the assertion that damage accumulation in bystander cells leads to stalled replication forks. As well as responses in bystander cells, DDR is also observed in cells where only the cytoplasm has been irradiated using microbeam approaches. Despite the historical dogma that DNA within the nucleus is the critical target, it is clear that cytoplasmic irradiation, involving mitochondrial responses, can lead to downstream biological consequences in terms of DDR, mutation formation cell killing, and apoptosis.

The Gray Cancer Institute has pioneered the use of X ray focussing techniques to develop systems for micro irradiating individual cells and sub cellular targets in vitro. Cellular micro irradiation is now recognised as a highly versatile technique for understanding how ionising radiation interacts with living cells and tissues. The strength of the technique lies in its ability to deliver precise doses of radiation to selected individual cells (or sub cellular targets). The application of this technique in the field of radiation biology continues to be of great interest for investigating a number of phenomena currently of concern to the radiobiological community. One important phenomenon is the so called ‘bystander effect’ where it is observed that unirradiated cells can also respond to signals transmitted by irradiated neighbours. Clearly, the ability of a microbeam to irradiate just a single cell or selected cells within a population is well suited to studying this effect. Our prototype ‘tabletop’ X-ray microprobe was optimised for focusing 278 eV C-K X rays and has been used successfully for a number of years. However, we have sought to develop a new variable energy soft X-ray microprobe capable of delivering focused CK (0.28 keV), Al-K (1.48 keV) andmore » notably, Ti-K (4.5 keV) X rays. Ti-K X rays are capable of penetrating several cell layers and are therefore much better suited to studies involving tissues and multi cellular layers. In our new design, X-rays are generated by the focussed electron bombardment of a material whose characteristic-K radiation is required. The source is mounted on a 1.5 x 1.0 metre optical table. Electrons are generated by a custom built gun, designed to operate up to 15 kV. The electrons are focused using a permanent neodymium iron boron magnet assembly. Focusing is achieved by adjusting the accelerating voltage and by fine tuning the target position via a vacuum position feedthrough. To analyze the electron beam properties, a custom built microscope is used to image the focussed beam on the target, through a vacuum window. The X-rays are focussed by a zone plate optical assembly mounted to the end of a hollow vertical tube that can be precisely positioned above the X ray source. The cell finding and positioning stage comprises an epi-fluorescence microscope and a feedback controlled 3 axis cell positioning stage, also mounted on the optical table. Independent vertical micro positioning of the microscope objective turret allows the focus of the microscope and the X ray focus to coincide in space (i.e. at the point where the cell should be positioned for exposure). The whole microscope stage assembly can be precisely raised or lowered, to cater for large differences in the focal length of the X ray zone plates. The facility is controlled by PC and the software provides full status and control of the source and makes use of a dual-screen for control and display during the automated cell finding and irradiation procedures.« less

Although conclusive evidence has been obtained for the presence of radiation-induced bystander effects, the mechanisms that trigger and regulate these processes are still largely unknown. The bystander effect may play a critical role in determining the biological effectiveness of low-dose exposures, but questions on how to incorporate it into current models and extrapolate the risks of radiation-induced carcinogenesis are still open. The Gray Cancer Institute soft X-ray microbeam has been used to investigate the dose-response relationship of the bystander effect below 0.5 Gy. The survival response of V79 cells was assessed after the irradiation of a single cell within a population with a submicrometer-size beam of carbon K X rays (278 eV). Above 0.3 Gy, the measured bystander cell killing was in agreement with previously published data; however, a significant increase in the scatter of the data was observed in the low-dose region (0.3 Gy). The data distribution observed indicates a binary behavior for triggering of the bystander response. According to our hypothesis, the probability of triggering a bystander response increases approximately linearly with the dose delivered to the single selected cell, reaching 100% above about 0.3 Gy. The magnitude of the bystander effect, when triggered, is approximately constant with the dose and results in an overall approximately 10% reduction in survival in our system. This suggests that the event that triggers the emission of the bystander signal by the hit cell is an all-or-nothing process. Extrapolation of the data indicates that when a single fast electron traverses a V79 cell, there is a probability of approximately 0.3% that the cell will emit the bystander signal. The data presented in this paper have also been analyzed statistically to test the possibility that complex DNA double-strand breaks may be the initial critical event.

The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 microm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0-2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (200 mGy). In the low-dose region (

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

Cellular micro-irradiation is now seen as a potent method for understanding how radiations interact with living cells and tissues. The strength of this technique lies in its ability to deliver precise doses of radiation to selected individual cells in vitro, or to pre-selected targets within cells. We have recently developed a focused soft X-ray microprobe for targeting individual cells. The use of focused X-rays for this type of study is unique, and is being applied in a number of novel experiments. One important application is to study the so-called bystander effect where un-irradiated cells are seen to respond to signals from nearby irradiated cells. It is also being used as a sub-cellular probe to compare the effects of nuclear versus cytoplasmic targeting. Our facility uses a 0.4-0.8 mm diameter zone plate to focus soft X-rays to a sub-micron beam. This is then aimed at selected sub-cellular targets using rapid automated cell finding and alignment procedures. The zone plate images characteristic-K X-rays of carbon or aluminium, generated by focusing a beam of 5-10 keV electrons on to the appropriate target. The current arrangement will deliver about 10000 photons/sec to the focus (sufficient to irradiate several tens of cells per minute).© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Charged-particle microbeams provide a unique is timely, as modern facilities will bene® t considerably opportunity to control precisely, the dose to individual cells and from some of the technological advances that have the localization of dose within the cell. The Gray Laboratory is occurred in recent years (for example, the use of now routinely operating a charged-particle microbeam capable computers in imaging, control of instrumentation of delivering targeted and counted particles to individual cells, and data acquisition systems). Furthermore, a at a dose-rate su ae cient to permit a number of single-cell assays of radiation damage to be implemented. By this means, it is number of recently developed or established biolo- possible to study a number of important radiobiological processes gical assays of radiation damage are now sensitive in ways that cannot be achieved using conventional methods. enough to be used at low doses, where microbeams This report describes the rationale, development and current will have a critical role. capabilities of the Gray Laboratory microbeam. This publication is the ® rst of a series to report on the design, implementation and use of the Gray

The use of a charged-particle microbeam provides a unique opportunity to control precisely, the number of particles traversing individual cells and the localization of dose within the cell. The accuracy of 'aiming' and of delivering a precise number of particles crucially depends on the design and implementation of the collimation and detection system. This report describes the methods available for collimating and detecting energetic particles in the context of a radiobiological microbeam. The arrangement developed at the Gray Laboratory uses either a 'V'-groove or a thick-walled glass capillary to achieve 2-5 microns spatial resolution. The particle detection system uses an 18 microns thick transmission scintillator and photomultiplier tube to detect particles with > 99% efficiency.

Cell survival was determined by clonogenic assay in human prostate cancer (DU145) and transformed fibroblast (AG0-1522) cells following exposure to modulated radiation fields created delivered using a X-Rad 225 kV X-ray generator. Uniform survival responses were compared to inand out-of-field responses in which 50% of the cell population was placed outside of the radiation field. Cell survival responses were determined under oxic and hypoxic conditions (0.1 % N2) and differential mechanisms of DNA damage response investigated use specific inhibitors of the DNA damage response pathway including ataxia telangiectasia mutated kinase (ATM, KU55933, Kudos Pharmaceuticals) and ataxia telangiectasia and Rad3-related protein kinase (ATR, Compound 45, Dr Chris Bakkenist, University of Pittsburgh). Temporal dependency of survival responses was determined by reseeding cells from the inand outof-field regions at different time post after irradiation. Differential mechanisms of cellular radiobiological response following exposure to intensity-modulated radiation fields

Uncertainties surround the radiobiological consequences of exposure to charged particles, despite the increasing use of accelerated ion beams for cancer treatment (hadrontherapy). In particular, little is known about the long-term effects on normal tissue at the beam entrance or in the distal part of the Spread-Out Bragg Peak (SOBP). Moreover, although the relative biological effectiveness (RBE) of particle radiation has been traditionally related to the radiation linear energy transfer (LET), it has become increasingly evident that radiation-induced cell death, as well as long term radiation effects, is not adequately described by this parameter. Hence, exploring the effectiveness of various ion beams at or around the Bragg peak of monoenergetic ion beams can prove useful to gain insights into the role played by parameters other than the particle LET in determining the outcome of particle radiation exposures. In this context, the upgrade of the Tandem irradiation facility at Naples University here described, has allowed us to perform a series of preliminary radiobiological measurements using proton and carbon ion beams. The facility is currently used to irradiate normal and cancer cell lines with ion beams such as oxygen and fluorine.

The X ray microprobe developed at the Gray Laboratory was originally designed to produce carbon K X rays (278 eV) by electron bombardment and focus them to a few hundred nanometers spot by using a circular diffraction grating with increasing line density (zone plate). The very fine focus achieved (< 0.25 micron radius spot) and the highly localised energy deposition of CK X rays (photoelectron range < 7 nm), represent unique tools to investigate modern radiobiological phenomena. Recent improvements have been directed to increase the dose rate (up to approximately 6 Gy.s-1 entrance dose averaged over a typical V79 cell) and to evaluate the possibility of using higher energy photons (AlK of 1.48 keV). The efficiency of the microprobe system has been tested by assessing the clonogenic potential of V79 cells irradiated with CK X ray beams of different sizes (5 and 0.25 microns radius) and investigating the relevance of the spatial distribution of cells for the bystander effect.