Investigation of the XUV Emission from the Interaction of Intense Femtosecond Laser Pulses with Solid Targets

The generation of coherent high-order harmonics from the interaction of ultra-intense femtosecond laser pulses with solid density plasmas holds the promise for table-top sources of intense extreme ultraviolet (XUV) and soft x-ray (SXR) radiation. Furthermore, they give rise to the prospect of combining the attosecond pulse duration of conventional gas-harmonic sources with the photon flux currently only available from large-scale free-electron laser or synchrotron facilities. In this thesis a series of experiments studying various aspects of harmonic generation from such a plasma source are presented and the emitted XUV-radiation is characterized spectrally, spatially and temporally. The measurements probe the dynamics of the plasma surface on a sub-laser-cycle time scale and help to increase our understanding of the harmonic generation process. It is shown that, at moderate intensities and laser contrast, the emitted harmonics are indeed phase-locked but chirped and emitted as a train of XUV-bursts of attosecond duration. Measurements with very high contrast relativistically intense driving pulses reveal the generation of harmonics up to the relativistic cutoff in a diffraction-limited beam with constant divergence observed for all wavelength. This implies that the harmonics are generated on a curved surface and travel through a focus after the target possibly opening a route towards extreme intensities in the process. In addition it is found that a target roughness on the scale of the wavelength of the highest generated harmonic does not adversely affect the harmonic beam quality implying that the generation of diffraction-limited keV-harmonic beams should be possible. In a third set of experiments the first demonstration of harmonic generation from solid targets using an 8 fs driving laser opens a route towards the generation of ultra-intense single-as pulses and gives conclusive evidence for the unequal spacing of the harmonic emission. Based on these results the development of ultra-intense sources of single as-pulses from the interaction of intense laser pulses with solid surfaces could advance at a fast pace making XUV-pump XUV-probe type investigations of nonlinear processes with attosecond time resolution feasible in the near future

Characterization of bistable states in rotating MBX mirror plasmas

In this thesis I report the observation and characterization of bifurcations and bistable states in both AC and DC biased rotating axis-symmetric mirror plasmas in the Magneto Bernoulli eXperiment (MBX). Several control parameters that characterize the bifurcations and bistabilities are identified and their influence on the rotational states is analyzed. The reaction of the plasma to changes in the identified control parameters is shown to be highly nonlinear and asymmetric. Evidence for the asymmetric response of rotating mirror plasmas to the polarity of the applied biasing voltage leading to good potential penetration at positive and bad penetration at negative biasing voltages that was shown before by Quevedo et al. [13] is given.Physic

Activation of an Antiviral Response in Normal but Not Transformed Mouse Cells: a New Determinant of Minute Virus of Mice Oncotropism ▿

Parvovirus minute virus of mice (MVMp) is endowed with oncotropic properties so far ascribed only to the dependency of the virus life cycle on cellular factors expressed during S phase and/or modulated by malignant transformation. For other viruses oncotropism relies on their inability to circumvent type I interferon (IFN)-induced innate antiviral mechanisms, the first line of defense triggered by normal cells against viral infections. These agents propagate, therefore, preferentially in transformed/tumor cells, which often lack functional antiviral mechanisms. The present study aimed at investigating whether antiviral processes also contribute to MVMp oncotropism. Our results demonstrate that in contrast to MVMp-permissive transformed mouse A9 fibroblasts, freshly isolated normal counterparts (mouse embryonic fibroblasts [MEFs]) mount, through production and release of type I IFNs upon their infection, an antiviral response against MVMp lytic multiplication. Pretreatment of MEFs with a type I IFN-β-neutralizing antibody, prior to MVMp infection, inhibits the virus-triggered antiviral response and improves the fulfillment of the MVMp life cycle. Our results also show that part of the A9 permissiveness to MVMp relies on the inability to produce type I IFNs upon parvovirus infection, a feature related either to an A9 intrinsic deficiency of this process or to an MVMp-triggered inhibitory mechanism, since stimulation of these cells by exogenous IFN-β strongly inhibits the parvovirus life cycle. Taken together, our results demonstrate for the first time that parvovirus infection triggers an innate antiviral response in normal cells and suggest that the MVMp oncotropism depends at least in part on the failure of infected transformed cells to mount such a response

TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

<div><p>The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9^+/+), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.</p> </div

Time-dependent transcriptional up-regulation of type-I IFN encoding mRNAs in MVMp- or H-1PV-infected human cell lines.

<p>(<b>A</b>) HEK293 and HEK293T, (<b>B</b>) NB324K and (<b>C</b>) Hela cells were mock-treated for 24 hrs, infected with parvoviruses (5 PFUs/cell) for the indicated times, or infected with NDV (6 HU/10⁶ cells) or transfected with pI:C (2 µg/ml) for 15 hrs. At the indicated times, cells were harvested and total RNAs were extracted using the RNeasy kit. One µg was then reverse transcribed into cDNA and 10% of this product was subjected to PCR reactions using sets of primers specific to each indicated cytokine mRNA or viral transcripts. PCR product of 18S ribosomal RNA was used as housekeeping gene to normalize loading. No signal was detected in the samples when omitting the reverse transcriptase. Presented data are representative of 3 experiments which all gave similar results.</p

Sensitivity to neuraminidase pre-treatment of the antiviral response triggered by parvoviruses in hPBMCs.

<p>(<b>A</b> to <b>C</b>) hPBMCs collected from the blood of healthy donors were distributed into 6-well plates at 1×10⁷ cells/5 ml culture medium/well. They were then treated or not with neuraminidase at 0.1 U/ml for 15 hrs and then mock-treated or infected with MVMp or H-1PV at 20 PFUs/cell. (<b>B</b>) In parallel to the treatment of PBMCs, HEK293 cells cultivated in 10-cm dishes at a density of 1.5 10⁶ cells/dish were also mock-treated or infected with MVMp or H-1PV at 2 PFUs/cell. Cells (<b>A</b>, <b>B</b>) as well as supernatants (<b>C</b>) were isolated 24 hrs p.i. in order to perform Southern blot (<b>A</b>), RT-PCR (<b>B</b>), and ELISA (<b>C</b>) experiments. (<b>A</b>) DNA was extracted from hPBMCs and Southern blotting performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">Figure 2</a>. (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded genome). The blot shown is representative of 3 experiments which all gave similar results. (<b>B</b>) Total RNA extraction and consecutive synthesis of cDNA from hPBMC and HEK293 cultures were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g001" target="_blank">Figure 1</a>. Expression of the indicated transcripts was assessed using specific pairs of primers. Transcripts encoding the human 18S ribosomal protein were used as internal loading controls. Data shown are representative of 3 experiments that gave similar results. (<b>C</b>) Collected supernatants were centrifuged in order to discard cellular debris and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in IFN-α and IFN-β cytokines. Results are expressed as means+standard deviations of three independent experiments. Each presented blot is representative of 3 experiments which gave similar results.</p

Time- and MOI-dependent production and release of type-I IFNs from parvovirus-infected hPBMCs.

<p>hPBMCs collected from the blood of healthy donors were distributed into 24-well plates at 1×10⁶ cells/500 µl culture medium/well. They were then mock-treated or infected with MVMp or H-1PV at 1, 10 or 50 PFUs/cell. After a period of incubation of 15, 24 or 48 hrs media were collected, centrifuged in order to discard cellular debris and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in IFN-α (<b>A</b>) and IFN-β (<b>B</b>). Results are expressed as means of three experiments.</p

Activation of a TLR-9 dependent production of type-I IFNs in parvovirus-infected human Namalwa cells.

<p>The cells (1.10⁶ cells/well of a 24-well plate) were either infected with MVMp or H-1PV (∼80 PFUs/cell equivalent to 1×10⁴ virus genomes/cell), stimulated with the TLR-9 agonist ODN 2395 at 1 µM, or mock-treated. (<b>A</b>) At the indicated time points culture supernatants were collected from the respective wells and used to determine the quantity of type-I IFNs released in the culture medium by ELISA experiments. Results are expressed as means+standard deviations of three independent experiments. (<b>B</b>) Mock-treated, parvovirus-infected or ODN stimulated Namalwa cells were harvested at the indicated time points and total RNAs were extracted using the RNeasy kit as described previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g001" target="_blank">Figure 1</a>. Total RNAs extracted from parvovirus-infected (24 hrs p.i. at 80 PFUs/cell) or pI:C-transfected (15 hrs post-transfection with 2 µg dsRNA/ml) NB324K cells were used as positive or negative controls. Presented data are representative of 3 experiments which all gave similar results. (<b>C</b>) Total DNA was harvested at the indicated time points from parvovirus-infected (MOI of 80 PFUs/cell) or mock-treated Namalwa or HEK293T cultures to assess by Southern blot experiments as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">Figure 2</a> the replication of both parvoviruses (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded DNA genome). The blot shown is representative of 3 experiments which gave similar results. (<b>D</b>) Cell pellets from mock-treated or parvovirus-infected Namalwa cultures were re-suspended in complete Ripa buffer and Western blotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g006" target="_blank">Figure 6</a>. Actin was used as an internal loading control. Each presented blot is representative of 3 experiments which gave similar results.</p

Assessment of a TLR-9-dependent activation of NF-κB in HEK-BlueTM-hTLR-9 cells upon MVMp or H-1PV infection.

<p>HEK-Blue™-hTLR-9 cells (8.10⁴ cells/well) cultivated for 24 hrs in 96-well plates following the manufacturer's instructions, were infected using increasing MOIs of MVMp or H-1PV for 24 hrs. NF-κB activity was then assessed by a colorimetric assay using a reporter system measuring the release of secreted embryonic alkaline phosphatase (SEAP) in the culture medium. Control experiments consisted in the stimulation of the latter cell line, pre-treated or not for 3 hrs with the TLR-9 inhibitor ODN TTAGGG at 2 µM, with the TLR-9 agonist ODN-2395 at 4 µM. Results are expressed as means+standard deviations of three independent experiments.</p