Four Month Report

THz-BRIDGE progress report February 1 to July 31, 2002 1

THz-BRIDGE progress report (Period from February 1 to July 31, 2003)

In the period from February 1 to July 31 – 2003, the spectroscopy activity of WP-1 has included a systematic characterization of amino-acids. The spectral features of saline solution for different valus of NaCl concentration have also been investigated and an effort has been made to get TD spectra of thin samples of blood constituents compared to serum and saline solution.

Irradiation studies have made a significant step forward with the completion of the Micronucleus assay on human lymphocytes in whole blood in the frequency band from 110 to 140 GHz and with the first results of the Comet assay on human lymphocytes, where the shielding effect of serum has been reduced as described in the following. Interesting results have also been obtained on the irradiation of lymphocytes cultures.

A database on the irradiation of epithelial cell cultures has been completed and a systematic investigation on the effects of 130 GHz exposure of liposomes at different modulation conditions has been carried out.

In the following, the progress of the activity at the various partner sites and on the individual work- packages of the project is described. The actions taken in the management of the project are also briefly described.

Project Management

The fifth project meeting was held at the University of Stuttgart, Germany on February 3-4, 2003 to discuss the progress of the research activity. The time schedule of the project and the relevant deliverables to be attached to the 2nd Year Report were also discussed. The Project Steering Committee (PSC) checked the annual Cost Statements to assure compliance with the EU contract regulations. The 2nd Year Report and all Cost Statements have been submnitted by the coordinator to the EU commission at the end of March. The Annual Report was approved at the end of June. The Cost Statement by the partner UFRANK was submitted without the signature of the Administrative Officer due to difficulties in the accounting of EU contracts by the UFRANK administration. At the end of July the problem was not yet solved and EU contract office decided in agreement with the co-ordinator to proceed with the payment to the other partners without any further delay if the signed UFRANK statement will not be submitted by mid of August.

Together with the Annual report, the co-ordinator submitted the Contract Amendment regarding the addition of the University of Freiburg (UFREI) as a new partner in the THz-BRIDGE consortium with an effective starting date of February 1, 2003. A revised table of distribution of costs was distributed to all partners. The Contract Amendment was finally received from Brussels and distributed to partners for signature at the beginning of July. The Amendment was sent back to the Commission for final signature at the end of July. Following an agreement with the partners UFRANK and UFREI a further request was also sent by he coordinator to the Commission to transfer 14.000 Euro from UFRANK to UFREI for the appointment of a post-doc.

Regarding the publication of the Proceedings of the THz-BRIDGE Workshop, as summarized at the meeting in Stuttgart, the whole package of the Proceedings was sent to the Editor Kluwer at the end of January. Proofs were distributed by Kluwer to the corresponding authors at the end of April. The publication of the Proceedings and distribution of the volume to registered participants occurred in June/July 2003. THz-BRIDGE progress report February 1 to July 31, 2002 2

The project web page: http://www.frascati.enea.it/THz-BRIDGE has been regularly maintained and updated at ENEA. The web page includes a public area, with general information on the project, and a password-protected area, with access restricted to the consortium partners. “Report-type” Deliverables are available on the public (PU) or restricted (RE) area of the web page according to their nature specified in the Deliverable List. Periodic progress reports have been made available to the public.

A spectral database of materials and biological systems at THz frequencies is being made available to the scientific community on the project web-page. Presentation material is available in electronic form in the reserved area of the project web-page.

THz Sources and Infrastructure Development ENEA  The microtron driven Compact FEL was used to perform irradiation of whole blood (se WP- 2.1). A new FEL source, FEL-CATS, is now operating in the spectral region between 0.4 and 0.7 THz and will be used for further irradiation studies.

FZR  Scientific activities at FZR have focused on the preparation and spectral characterisation of biological material in the 5-20 THz spectral range. A FTIR-spectrometer has been installed at the ELBE and equipped with optics and detectors for measurements down to 3 THz. We have identified absorption bands around 8 and 15 THz in lipids which will be further investigated in pump probe experiments using the ELBE FEL. A method has been established to investigate in the mid IR whole cells cultured in-house and allowing quick transfer to experimental stations. This method will be extended to allow absorption measurements below 20 THz.

 To circumvent problems with limited THz transmittance of certain materials FZR has started to set up a modular external reflection device to study supported biofilms in the THz-regime. The apparatus has been designed to allow maximal freedom in the choice of pump and probe wavelengths. In parallel, the construction of a matrix isolation system was continued and has been brought to near completion (all vacuum equipment installed and tested). It will be used to study intra- and intermolecular processes in small biological molecules by measuring their vibrational relaxation properties.

 The optical resonator for the ELBE FEL was mounted completely for the undulator U27 which will allow to asses the range below 20 THz. All mechanical equipment and optical components (alignment system, interferometer for length stabilization, mirror wheels, temperature stabilization of the mirrors) have been checked thoroughly and tested for long term stability. In order to perform two color pump-probe experiments the free-electron laser has to be synchronised with high precision with solid-state mode-locked femtosecond lasers. The achievable time resolution is limited by the pulse duration of the involved lasers and the timing jitter of the synchronization. FZR has synchronized a femtosecond mode-locked Ti:sapphire oscillator delivering 12 fs pulses at a repetition rate of 78 MHz. This frequency equals six times the anticipated repetition rate of the free-electron laser (FEL) of 13 MHz. The phase-locked loop of the synchronization is stabilized at the 30th harmonics of the FEL repetition rate (390 MHz). The timing jitter of the femtosecond oscillator has been determined from the noise THz-BRIDGE progress report February 1 to July 31, 2002 3

spectral density to be below 400 fs, which is better than values reported up to date in the literature.

WP-1 (Spectroscopy of proteins, enzymes, biological membranes, and selected cells)

In the period of this progress report, USTUTT progressed significantly in the collection of spectral data of amino acids. Measurements have followed a few directions: + - (1) characterization of glycine (NH3 CH2COO ) and attempt to assign the observed vibrational features, (2) characterization of glycine chains of increasing length: diglycine (C4H8N2O3), triglycine (C6H11N3O4), tetraglycine (C8H14N4O5), pentaglycine (C10H17N5O6), etc. (3) characterization of hybrid samples: AlaGly, LeuGly.

Glycine is one of the most studied amino acids; it is also the smallest from the standard amino acids. An analysis of the normal vibrations of glycine was done for the first time in 1949 by Baba et al. [1]. They approximated glycine as a five-atom system. A more detailed study to include the analysis of the normal vibrations of glycine as a seven-body system was conducted by Suzuki et al. [2]. Here were also included the absorption spectra of crystals of glycine, glycine-d3, glycine-d2 and -1 glycine-d5 between 280 and 4000 cm . Later, they performed identical measurements and calculations for the chains of these molecules [3]. In 1983 Genzel et al. [4] extended the measurement frequency range for glycine down to 20 cm-1.

USTUTT investigations on glycine spanned the spectral range between 50 and 670 cm-1. For 15 15 + - glycine and glycine- N ( NH3 CH2COO ); optical transmission experiments were performed at room temperatures and at temperatures as low as ~5K. The samples were prepared as pressed pellets mixed with polyethylene powder in the ratio 1:10. In Fig.1 the transmission spectra of the three different types of glycine are plotted for high and low temperatures.

Fig. 1 Frequency dependent transmission of glycine. The different spectra show the dependence on temperature and isotope for glycine and glycine-15N.

The resolution in the above measurements is 2 cm-1. THz-BRIDGE progress report February 1 to July 31, 2002 4

The spectra are rich in low-frequency vibrational bands. As the temperature decreases, these low- lying bands split and become sharper. We assume that the features lower than 200 cm -1 are due to the lattice modes associated with vibrations of hydrogen bonds. In case of glycine-14N and glycine- 15N the spectra show two broad peaks at 132 and 164 cm-1 at room temperature. Upon cooling, these peaks split in three maxima located at 123, 144 and 179 cm-1. As these peaks appear in both kinds of glycine, they must be determined by the lattice, that hardens upon cooling. Another peak due to the lattice vibrations is at 214 cm-1, found in both glycine-14N and glycine-15N at 5 K (fig. 2); at 300 K this peak is not visible. The amine group is involved in the vibration from 358/364 cm-1 (300/5K) found in glycine: in glycine-15N, this peak is shifted to 353/358 cm-1 (300/5 K).

Fig. 2 Gly 14N and Gly 15N at 5K. (spectra of Gly 14N is displaced up in order to observe how the minima shifts).

Observing the broad bands shown by glycine at room temperature, where neither sharp lines nor preferred absorption frequencies are present, one would not expect any damage of the sample at such frequencies

Another set of measurements we dedicated to Thz transmission measurement through glycine samples as a function of molecule size. For the identification of material composition through its specral data, it is necessary to have a monotone variation of the absorption with the sample mass. In this respect USTUTT performed measurements on glycine oligomers, of increasing chain length (fig.3). Similar measurements were performed earlier by Shotts et al. (5) . THz-BRIDGE progress report February 1 to July 31, 2002 5

Fig. 3 Temperature dependence of transmission of diglycine, triglycine, tetraglycine, and pentaglycine (pressed pellets, mixed with polyethylene, room temperature).

. We found that the absorption spectra show more features when going from one to two and three glycine molecules; however no simple analysis is possible since the features do not change monotonous from sample to sample, as the mass does. Our measurements were made at room temperature, but we expect that at low temperature situation is the same, with the difference that the spectra of smaller oligomers have sharper peaks than the bigger oligomers. This can be explained in terms of lattice structure: the smaller the oligomer, the better defined the crystal structure. When the oligomer is getting bigger, the crystals tend to be disordered, resulting in broader features.

Fig.4 Transmission of LeuGly and AlaGly , pressed pellets mixed with polyethylene, room temperature. THz-BRIDGE progress report February 1 to July 31, 2002 6

In the third set of measurements USTUTT investigated the behaviour of amino acids bound in hybrid molecules (like AlaGly and LeuGly). The transmission spectra of these samples are presented in the Fig.4. As for glycine oligomers, the objective was to see if the features from single amino acids are found in a molecule with two different amino acids. At a first inspection, this is not the case or at least the analysis is not straight foreward, the new molecule has a different spectra from the original two molecules. Another interesting issue is thesearch for any sharp features in the new spectrum, to serve as an indication for a weak spot in the crystalline lattice. The results are, yet, not completely analyzed.

[1] H. Baba, A. Mukai, T. Shimanouchi and S. Mizushima, Nippon Kagaku Zasshi 70, 333 (1949). [2] S. Suzuki, T. Shimanouchi and M. Tsuboi, Spectrochimica Acta, 19, 1195-1208 (1963) [3] S. Suzuki, Y. Iwashita, T. Shimanouchi and M. Tsuboi, Biopolymers, 4, 337-350 (1966) [4] L. Genzel, F. Kremer, A.Poglitsch, and G.Bechtold, Coherent Excitations in Biological Systems, Ed. H. Fröhlich and F. Kremer, Springer-Verlag Berlin Heidelberg (1983) [5] W.J. Shotts and A.J. Sievers, Biopolymers, 13, 2593-2614 (1974) THz-BRIDGE progress report February 1 to July 31, 2002 7

Terahertz Time Domain Spectroscopy (TDS) of biological materials

The investigation on the effects of salt concentration on the terahertz absorption spectrum of water has been completed at TVL. The contrast mechanism for biomedical imaging using terahertz radiation is still not fully understood. However, it is believed that it is a change of water content in tumour is the primary mechanism. To test if salt concentration had any importance to the terahertz image a number of sodium chloride solution were prepared. Figure 5 shows the spectrum of pure water and the spectra over a range of different concentrations (125 mM, 500 mM, 2 M and 4M) of sodium chloride (NaCl). Figure 6 shows the differences in refractive index. The physiological concentration of salt in the human body is approximately 150 mM. Since there is no modification to the terahertz absorption spectrum until non- physiological concentrations of salt are reached we can speculate that NaCl does not contribute as a possible contrast mechanism in a terahertz pulse image. The increase in absorption seen in Figure 5 is believed to be due the increase in electrical conductivity of the solution. The broad features in the spectra, at 0.6 and 1.2 THz, are due to an etalon effect from the z-cut quartz windows.

Fig. 5: Terahertz absorption spectra of Fig. 6: The refractive index of different concentrations of salt different concentrations of solution vary from pure water to 4M salt solution vary from pure THz-BRIDGE progress report February 1 to July 31, 2002 8

WP-2 : Evaluation of biological effects in vitro after exposure to THz-radiation WP-2.1 (DNA bases and human lymphocytes)

Irradiation of whole blood & effects on human lymphocytes The biological experimental conditions to be adopted, such as blood sampling, sterile containers to be used to expose samples, and the construction of THz delivery beam lines at ENEA were discussed in previous reports. According to the exposure protocol discussed in the previous reports, for the irradiation of human lymphocytes blood samples are taken from each donor using sterile Vacutainers. Samples are prepared (2 ml each) and put into polystyrene Petri dishes. In order to have a transmission through the blood sample sufficient to monitor the THz power during irradiation a dish with internal diameter of 52 mm is used, corresponding to about 900 m thickness of the sample. The irradiation of the first nine subjects has been described in the 1st and 2nd Annual Reports.

During the present progress period:  Blood samples from 10 healthy donors have been exposed/sham exposed at frequency of 130 GHz (ENEA, ICEmB) in order to complete the study devoted to investigate the induction of micronuclei following exposure to THz radiation. The exposure time was 20 minutes. Two different average power have been studied 3.5 mW and 5.0 mW with a pulse repetition rate of 5 and 7 Hz respectively. Lymphocyte cultures have been established by using exposed blood from 5 healthy donors for each exposure condition. Micronucleus frequency and cell cycle kinetics have been evaluated by applying the cytokinesis block micronucleus technique (ICEmB)  The methodology to apply the alkaline comet assay to exposed/sham exposed samples has been set up (ICEmB). Suitable human blood samples have been developed to be exposed to THz radiation in order to apply the comet assay.  Blood samples from 14 healthy donors have been exposed/sham exposed for 20 minutes to 130 GHz with a pulse repetition rate of 7 Hz to evaluate DNA damage by applying the alkaline Comet assay. Blood samples from 9 donors were exposed to an average power of 5.0 mW while blood samples from the remaining 5 donors were exposed to 1.9 mW.

Methods

Set up of blood samples to be exposed for the study of micronucleus induction

Blood samples from 10 healthy subjects (mean age of 37.8 ± 7.2 years) were collected at ENEA occupational health unit. For each blood sample, two Petri dishes were set-up containing 2 ml each: one to be exposed, the other to be sham exposed. From each of them a lymphocyte culture was set up, a control culture with no exposed/sham exposed blood was not set-up since, in the previous experiments, we demonstrated that no significant difference, in terms of MN frequency, was detected between the control cultures and the sham exposed ones. An aliquot of whole blood from 4 of the 10 subjects has also been analyzed for DNA damage by Comet assay, in order to verify the induction of genotoxic effects soon after the exposure/sham exposure.

Lymphocyte cultures and cytokinesis block

Whole blood cultures are set up by adding 0.8 ml whole blood to 9.2 ml of RPMI medium supplemented with 15 % heat-inactivated foetal calf serum, 2 mM L-Glutamine and 100 µl of Phytohemagglutinin as mitogen. In order to block cytokinesis, 44 hours after PHA stimulation Cytochalasin-B (2 mg/ml in DMSO) is added to cultures to give a final concentration of 6 µg/ml. 72 h after PHA stimulation, cells are collected and processed for slide preparation as follows: after gentle homogenisation of the cell suspension, cell culture is centrifuged and the cell pellet is THz-BRIDGE progress report February 1 to July 31, 2002 9

resuspended in 3 ml of lysis buffer (10 mM EDTA, 155 mM NH4Cl and 10 mM KHCO3) and left for 7 min at room temperature, to break erythrocytes. Lymphocytes are then washed three times in RPMI + 2% FCS (wash medium), swelled by a soft hypotonic solution (one part of wash medium and four parts of distilled water) for 15 min and spun down onto slides using a cytocentrifuge (Cytospin, Shandon; 7 min at 1200 rpm). After fixation (80% methanol in aqueous solution for 10 min) and staining (5% Giemsa in phosphate buffer, pH = 6.8 for 10 min) coded slides are scored blindly and MN are counted in binucleated cytokinesis-blocked cells with a light microscope at 1250 x magnification following standard criteria For each subject/treatment 1000 binucleated cells are examined from various slides.

Cell cycle kinetics

On the same slides prepared for MN scoring, cell proliferation is also evaluated by scoring the number of nuclei in 500 cells and determining the Cytokinesis-block proliferation index (CBPI): it measures the mean number of cell cycles per cell and is defined as follows: CBPI = [M1 + 2M2 + 3(M3 + M4)] / N where M1 to M4 indicate the number of cells with 1 to 4 nuclei respectively, and N the total number of cells scored.

Set up of blood samples to be exposed for the study of DNA damage by means of comet assay.

Blood samples from 10 healthy subjects ranging from 26 to 52 years (mean age of 39.3  8.74) were collected at ENEA occupational health unit. For each blood sample two eppendorf tubes (1.8 ml) containing each 1.5 ml of whole blood have been set up, one to be exposed and the other one to be sham exposed. Such samples have been centrifuged at 1000 rpm in order to separate the serum from the cellular portion of the blood, in order to reduce the liquid layer and optimized the exposure . A suitable portion of serum has been removed to avoid the aspiration of lymphocytes that lie at the interphase between the red blood cells, positioned at the bottom of the tube, and the serum. Following the exposure /sham exposure the homologous serum is added and the blood samples are processed to perform Comet assay according to the method developed by Singh and co-workers (Singh et al., 1988), with minor modifications .

Slide preparation

10 µl of whole blood are mixed with 65 l of low melting agarose ( LMA 0.7%, 37 °C) and rapidly pipetted onto 1% normal melting agarose (NMA) pre-coated slides, spread using a coverslip and maintained on a cold flat tray for about 5 min to solidify. After removal of the coverslip the third layer ( 75 l LMA 0.7% , 37°C) was added and solidified. After removing the coverslip the slides are immersed in freshly prepared cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA,10 mM Tris, pH 10 with 1% Triton X-100 and 10% DMSO ) and lysed overnight at 4°C. Subsequently, the slides are drained and placed in an horizontal gel electrphoresis tank, filled with fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH >13) and left in the solution for 25 min at 4°C, to allow the equilibration and the unwinding of DNA to express the alkali labile damage before electrophoresis. Electrophoresis was carried out at 4°C for 25 min, at 30 V (1 V/cm) and 300 mA, by using an Amersham Pharmacia Biotech power supply, and adjusting the current to 300 mA by modulating the buffer level. Following electrophoresis the slides are rinsed with sodium acetate (300 mM)/absolute ethanol (90%) solution for 30 min. and dehydrated in absolute ethanol for 2 h, then the slides are rehydrated in 95% ethanol for 5 min. THz-BRIDGE progress report February 1 to July 31, 2002 10

Slide scoring and data analysis

Slides were stained, just before analysis, with 60 l of ethidium bromide (12 g/ml) and images of 100 randomly selected cells (50 from each of two replicate slides) were analyzed, from each sample, by a computerized Image Analysis System (Delta Sistemi, Rome, Italy). This system acquires images, computer the integrated intensity profile for each cell, estimate the comet cell components, head and tail, and evaluate a range of derived parameters. The 100 DNA spots are classified into five categories corresponding to the amount of DNA in the tail according to Anderson et al. Results are expressed as “comet tail factor” calculated according to Ivancsits et al. : tail factor (%) = [ AFA + BFB + CFC + DFD + EFE ] / 100 where A is the number of cells classified to group A, FA the average of group A, B is the number of cells classified to group B, FB the average of group B, C is the number of cells classified to group C, FC the average of group C, D is the number of cells classified to group D, FD the average of group D, E is the number of cells classified to group E, FE the average of group E.

Statistics

In order to compare each exposed sample with its sham exposed control, two tailed paired Student’s t test is applied for MN frequency, CBPI and % tail factor. Differences are considered to be significant at P<0.05.

RESULTS

Exposure of human blood to 130 GHz THz radiation for the evaluation of micronucleus frequency and CBPI

The following irradiation parameters have been obtained for the first group of 5 donors.

Frequency Bunch Bunch Pulse Pulse Average Irradiatio Delivered duration interval duration rep. rate power n time energy 130 GHz 50 ps 330 ps 4 s 5 Hz 3.5 mW 20 min. 4.20 J

Table 1: irradiaton parameters for the first group of donors

The results obtained following 20 min. exposure are depicted in Fig. 7 for both micronucleus frequency and CBPI. THz-BRIDGE progress report February 1 to July 31, 2002 11

0,7 2 %MN

) 0,6 %

( 1,5 CBPI 0,5 y c I n

0,4 P e u 1 B q C

e 0,3 r F 0,2 N 0,5 M 0,1 0 0 Sham Exposed Sham Exposed

Figure 7 – Micronucleus frequency (expressed as percentage) and CBPI in lymphocyte cultures from sham exposed and 130 GHz exposed blood samples from 5 donors. Data are reported as mean  SD.

The following irradiation parameters have been obtained for the second group of 5 donors.

Table 2: irradiaton parameters for the second group of donors

The results obtained following 20 min. exposure are depicted in Fig. 8 for both micronucleus frequency and CBPI, while the results obtained in terms of DNA damage by evaluating the % tail factor are reported in Fig. 9.

%MN 0,7 2 CBPI 0,6 )

n 0,4 P e B

u 1 q 0,3 C e r F 0,2 N 0,5 M 0,1 0 0 Sham Exposed Sham Exposed

Figure 8 – Micronucleus frequency (expressed as percentage) and CBPI in lymphocyte cultures from sham exposed and 130 GHz exposed blood samples from 5 donors. Data are reported as mean  SD.

EXP r o t 3 SH c a

0 Treatments THz-BRIDGE progress report February 1 to July 31, 2002 12

Figure 9 – Alkaline comet assay tail factor (%) of 130 GHz exposed blood sample from 4 healthy donors. Data are reported as mean  S.E.

Exposure of human blood to 130 GHz THz radiation for the evaluation of DNA damage (Comet assay)

The following irradiation parameters have been obtained for the first group of 5 donors and the results are reported in figure 10.

Table 3: irradiaton parameters for the first group of donors

6 5 ) % (

o EXP t

i 2 a T 1 0 Treatments

Figure 10 - Alkaline comet assay tail factor (%) of 130 GHz exposed blood sample from 5 healthy donors. Data are reported as mean  S.E.

The following irradiation parameters have been obtained for the second group of 5 donors and the results are reported in Fig. 11.

Table 4: irradiaton parameters for the second group of donors THz-BRIDGE progress report February 1 to July 31, 2002 13

) 4 % (

o 3 EXP t c a

f 2 SH

l i a 1 T 0 Treatm ents

Figure 11 - Alkaline comet assay tail factor (%) of 130 GHz exposed blood sample from 5 healthy donors. Data are reported as mean  S.E.

Conclusions From the analysis of the results obtained, it appears that no genotoxic effects occur following 130 GHz THz exposure of whole blood samples from healthy subjects. This result has been obtained in different irradiation conditions by evaluating the micronucleus frequency, and has been confirmed by applying the comet assay. A statistically significant increase in tail factor has been detected when 1.9 mW average power and 2.28 J of delivered energy, with a pulse repetition rate of 7 Hz have been compared with sham exposed samples (P = 0.013), indicating an increase in DNA strand breaks in human lymphocytes. On the other hand, it is interesting to note that when human blood is treated with a well known comet inducer (Methyl methane sulphonate, MMS) a 6 fold increase has been detected (tail factor % = 5% vs. 30% in control and MMS treated samples, respectively). In order to better clarify the increase found in the 5 subjects studied, further investigation are needed in this experimental condition. Concerning the cell cycle kinetics it also resulted unaffected in all the exposure condition adopted as stated by analyzing CBPI.

References

D. Anderson, T.W. Yu, B. J. Phillips, P. Schmerzer. The effects of various antioxidants and other modifyed agents on oxigen radical generated DNA damage in human lymphocytes in the comet assay, Mutation Research, 307 261-271 (1993).

S. Ivancsits , E. Diem, A. Pilger, H.W. Rudiger , O. Jahn, Induction of DNA strand breaks by intermitted exposure to extremely low frequency electromagnetic fields in human diploid fibroblasts. Mutation Research, 519 1-13 (2002). THz-BRIDGE progress report February 1 to July 31, 2002 14

Irradiation studies at TAU

Modification of the exposure system Due to the detection of some mechanical vibrations in the previous constructed exposure system and in order to improve temperature stability the exposure chamber was replaced by a new one based on a commercial type incubator (Model BD115, Binder, Germany). The walls of the incubator were covered with microwave absorbing material, apart from the bottom, to prevent reflections and scattering of the radiation beam from the walls.

Assessment of radiation effects Following the comments of the mid-term review, TAU exposed lymphocytes isolated from human peripheral blood by ficoll gradient. Samples taken from three individuals were exposed for 1, 2 and 24 hours.

Analysis In order to enhance both the throughput and the reliability of the aneuploidy measurements TAU employed a recently acquired image analysis station. It is composed of an automatized Zeiss microscope with a software (Metacyte Fish Spot Counting), purchased from Metaphase Systems (Germany) to carry out automatized analysis of aneuploidy. For the analysis of aneuploidy, 600 interphase cells were scored for the number of signals representing the number of chromosome in those cells. The total level of aneuploidy is given as the proportion of cells with less then the expected two signals (monosomy) or more then the expected two (multisomy). For the analysis of replication timing and asynchrony, 600 interphase cells with two hybridization signals were examined. The shape of the signal was noted and recorded as previously described. In brief, signals were marked as representing unreplicated sequence (single spots, S) or replicated sequences (double dots, D). Each cell was scored as either early replicating (having two already replicated sequences, DD), as late replicating (having two as yet unreplicated sequences, SS) or as an asynchronous cell (having one replicated and one as yet unreplicated sequence, SD). The proportion of cells in each category was noted. TAU carried out a dual FISH labeling of all cells with DNA probes for centromers 11 and 17. The aneuploidy of chromosomes 11 and 17 was carried out simultaneously in the same cells. Similarly, the replication patterns of both 11 and 17 centromers was also carried out simultaneously in the same cells.

Results Analysis of the two hours exposed samples to 100GHz of three individuals (Fig. 12), reveals statistically significant changes in the level aneuploidy (comparison of exposed. vs. sham of chromosomes 17 and 11 yields p values of 0.0034 and 0.0007 respectively).

ANEUPLOIDY 18 16 14 12 10 8 6 4 2 ave 17 11

Figure 12 Percent of aneuploidy of chromosome 17 and chromosome 11 in human lymphocytes exposed to 100 GHz radiation for 2 hours. The samples are given in terms of mean  standard deviation of samples of three individuals, where each individual was scored for 600 cells. Blue –exposed; Brown –sham; Yellow –control. THz-BRIDGE progress report February 1 to July 31, 2002 15

Analysis of the replication patterns (Fig. 13) reveals a statistically increase in the anomalous asynchronous replication (SD) of both 17 and 11 centromers following exposure as compared to sham or controls (comparison of exposed. vs. sham of chromosomes 17 and 11 yields p values of 1.6x10-6 and 2.3x10-6, respectively ). The increase in the SD fraction following exposures seems to be at the expense of reduction of the SS fraction (reflecting late replication).

SS SD DD

35.00 90.00 8.00 7.00 85.00 30.00 6.00 80.00 25.00 5.00 4.00 75.00 20.00 3.00

70.00 15.00 2.00 1.00 65.00 10.00 0.00 17 11 17 11 17 11 exp sham control exp sham control exp sham control

Figure 13: Percent of SS, DD and SD patterns of replications of centromers 17 and 11 of human lymphocytes exposed to 100GHz radiation for 2 hours. The samples are given in terms of mean  standart deviation of samples of three individuals, where each individual was scored for 600 cells. Blue –exposed; Brown –sham; Yellow –control.

The results obtained clearly demonstrate that the above exposure conditions can elicit genomic instability in human lymphocytes.

THz irradiation of DNA and RNA bases. Taking into consideration the experimental results obtained so far by NHRF and described in the Annual Reports, and the fact that no photochemical damage was observed on DNA bases at long wavelengths, a more refined technique will be employed to detect any DNA potential damage by applying Ultra High Resolution Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy (fig. 14), of illuminated samples at THz frequencies. A similar method was previously applied for the investigation of structural damages in biological macromolecules at shorter wavelengths. For the last six months samples exposed to THz radiation at ENEA were analyzed with high resolution microscopy to assess possible potential damage by comparison with structural damages at 157nm. Results are still not conclusive, more experiments of this kind are required to be carried out with controllable radiation parameters.

Figure 14: AFM image of Guanine following its exposure to at THz radiation at ENEA. THz-BRIDGE progress report February 1 to July 31, 2002 16

WP-2.2 : Evaluation of biological effects in vitro (and Epithelial cell cultures and Membranes)

Studies with epithelial cells Following the MidTerm Review comments by the referees concerning Workpackage 2.2 and this aspect of the study, a number of changes were made to the basic study plan in line with their recommendations. 1) We have begun to evaluate the potential effects of co-cultures of human epidermal cells and neural cells. 2) We have begun the evaluation of the effects of THz radiation on dividing keratinocytes since these are going to be the most sensitive keratinocyte population. 3) We have begun the evaluation of THz radiation effects upon neural cells, in particular the ND7/23 sensory neuronal cell line. 4) We have exposed cells on the machine from Teraveiw that has been used in human studies of THz imaging as well as more powerful sources at Leeds University.

As outlined in the previous report for February 2002 – January 2003 under “Approach for Final Year,” we have begun the examination of the effects of THz radiation on dividing epithelial cells and their subsequent ability to differentiate. Two approaches have been applied. a) The cells were exposed when dividing, then allowed to grow to confluence prior to being differentiated.

The reason for this approach is that it does correspond with the previous approaches with confluent cells so that comparisons can be made. This was undertaken when the Teraveiw portable source was in the laboratory. TVL loaned University of Nottingham the TPI scan for a period of two weeks, starting 24th, February 2003. The instrument was supported by Dr V. Wallace (TVL) and Miss E. Pickwell (University of Cambridge).

Method The normal human keratinocytes were seeded onto a 96 well plate in clusters of four cells. Then 4 wells were exposed at each of three time points with the height of the plate from the source being adjusted such that the beam was focused on the cells attached to the bottom of the wells. The cells were then returned to normal medium culture conditions, grown on to confluence and then driven to differentiate in the presence of the fluorescein cadaverine.

Results No evidence of significant changes in ability to grow or differentiate was observed.

Conclusions a) Since an exposure time of over twice that maximally employed with human patients was examined, and since the dividing cells were directly exposed i.e. without the normal stratum corneum, granulosum and spinosum in human skin, it is concluded that no damage was detectable with this source. THz-BRIDGE progress report February 1 to July 31, 2002 17

b) The cells were exposed to THz as above and also using a more powerful source at Leeds. The cells were then immediately stimulated to differentiate.

The reason for these changes was to check that during the time the dividing keratinocytes were cultured after exposure, they were not recovering from the damage. However, since we were also requested to test more powerful sources and thus needed to transport the cells to these sources, Leeds University was initially tried. Leeds rather than Teraview in that they are half an hour closer in traveling time, thus in total 1 hour is cut off the time the cells are out of the normal culture conditions, being 5hrs instead of 6.5hrs.

Method The cells were plated as for the previous experiment. The transportation box was heated to 37°C and a heating mat was also included. The cells were exposed to a focused beam, as before, to cover the 4 well cluster with the heating mat applied just out of the beam in an attempt to keep the temperature up in the cooled rooms required by the lasers. The cells were exposed for a 45 minute fixed period. The cells were returned assayed for activity and then cultured in the fluorescein cadaverine containing differentiation medium.

Results No adverse effects were noted.

When tried with the Teraview portable source in Nottingham there was no sign of any adverse effects on growth or differentiation. However, the source had to be returned to Cambridge and thus repeat experiments have not yet been possible. Negotiations for another period for the machine in Nottingham are underway. The Leeds experiments have run into problems. The laser room temperatures are 21 – 23°C and while the heating mat can keep the cells at 25-28°C they clearly suffer as a result of this drop in temperature during the 45 minute and the 1.5hrs travel to and from Leeds. Thus we have had problems with culturing the epithelial cells for an extended period of 9 days (as usual) after the exposure. We are not sure how to resolve this transportation temperature problem to which dividing keratinocytes are clearly sensitive. We have some ideas about heating but the lack of CO2 buffering may also compound the problem.

Conclusions We also indicated following the mid term review, that we planned to look at the ND7/23 cells in co- culture with the keratinocytes.

Effects of THz on ND7/23 cells We have conducted a number of experiments in parallel with those on keratinocytes, given above. a) The effects of THz on neural cells and their activity and ability to take in fluorescein cadaverin following THz exposure. The neural cells seem to be more robust, probably since they are a cell line.

Methods The same approaches given above for keratinocytes were also examined with the ND7/23 cells. Thus clusters of 4 wells were exposed in the same way then differentiated with the keratinocyte medium containing fluorescein cadaverin. THz-BRIDGE progress report February 1 to July 31, 2002 18

Results The ND7/23 cells survived the travel and exposure better than the keratinocytes and showed no real change in FC uptake of cell activity over the 8 days in culture following exposure.

Conclusion The differentiation capacity and cell activity of the neural cells is not affected by the THz levels employed.

Effects on ND7/23 co-cultures. b) Cells grown together in the same wells This has been attempted once to ascertain if there would be an adverse effects when the cells were grown together in the same wells. Method ND7/23 cells are seeded into the wells and then once they have attached for 2 hours the keratinocytes were added. Control keratinocytes alone and ND7/23 cells alone were also set up. Thus in a 4 well cluster there was on with NHK cells one with ND7/23 and two with co-cultures. The medium was changed to differentiation medium directly after the exposure and then assayed on day 3, 9, and 12 post exposure. Results No adverse effects were noted. We have only been able to do this once, but hope to repeat it when the portable source can be loaned again.

c) A complex keratinocytes / ND7/23 co-culture exposure. This has only been attempted once to date. In this approach the portable source is required because of the nature of the exposure.

Method ND7/23 cells are seeded onto the underside of an insert allowed to attach and then the insert is turned the correct way up and keratinocytes seeded onto the top surface. Thus the keratinocytes are on one side of the porous insert and the neural cells on the other, i.e. the cell bodies are physically separated as in vivo.

Since the THz would penetrate the keratinocytes before reaching the neural cells in the dermis below in vivo, in the in vitro model the insert needed to be inverted into the THz beam such that it was focused on the keratinocytes.

Thus, the inserts were transferred onto the lid of a 24 well plate, the base of the plate added to retain humidity whilst the cultures was exposed. Since 24 well inserts are used only one insert at a time could be exposed.

Single inserts with NHK cells or ND7/23 cells were exposed and one coculture was exposed via the NHK cell surface, the other via the ND7/23 cell surface. After exposure the cells were cultured for 1 day then a resazurin and fluorescein leakage assay performed to check for barrier function integrity. The medium was changed to differentiation medium and the inserts assayed two days later for activity and FC incorporation

Results We have only been able to do this once, but hope to repeat it when the portable source can be loaned again. THz-BRIDGE progress report February 1 to July 31, 2002 19

However, it has demonstrated that this approach is possible, the cells do survive and can be returned to assess growth and differentiation. Also, it is possible to either exposure the keratinocytes or the neural cells. Also the barrier function is retained with far less than the maximal acceptable leakage of 6% occurring.

Further work For the complex co-culture models we are dependant upon the THz source being next to tissue culture facilities such that the co-cultures are returned to medium and 37°C as soon as possible after exposure. As for the effects upon dividing cells, we are trying to look at is how to keep the cells warm in transit. Also to test effects on cells with photoactivatable chemicals in them, as originally outlined. Other studies on the ND7/23 cells have shown that the cell tracker green and chromogranin A antibodies do not clearly distinguish differentiated from non-differentiated neural cells. So in associated projects not funded by the EU, this issue is being addressed.

THz Exposure of CA-loaded Liposomes

Irradiation set-up Two different irradiation set-up have been designed and constructed, referred to as A (Fig. 15) and B (Fig. 16).

A) The FEL radiation at the central frequency of 130 GHz is transported through a copper light-pipe of 25 mm diameter and is then fed into a “THz delivery system” (TDS) specifically designed to match the above requirements. The TDS is made of aluminum and is built to provide a uniform irradiation of the liposome samples.

Figure 15: Setup A

The THz beam coming from the light pipe into the TDS is first focused down to a 17.5 mm diameter aperture by means of a conical section and is then let expand by diffraction to about 52 mm diameter to match the required irradiation area. Calculations have been performed in order to minimize internal reflections in the cone, making the beam expansion similar to the free space expansion. The cuvette containing the liposome samples are placed in a Teflon grid, positioned in the region of maximum uniformity of the field in the TDS over the entire time of exposures (60 min). At the end THz-BRIDGE progress report February 1 to July 31, 2002 20 of exposures the kinetic measurements are immediately carried out.

B) In order to irradiate the liposomes and carry out the kinetic measurements in real time, the cell compartment of the spectrophotometer has been modified to deliver the THz beam onto the cuvette containing the liposomes in a final volume of 3 ml. Two possible irradiation geometries have been considered as described in the deliverable D-9a (Fig. 16 and Fig. 17). In the present experiments we used an irradiation from the side of the cuvette. An electroformed copper horn is used to match the circular cross section of the THz beam to the rectangular cross section of the cuvette.

Figure 16: Cell-beam spectrophotometer modification:irradiation from the side of the cuvette.

SAMPLE ION IAT RAD

Figure 17: Setup B

Experimental Procedure We recall the procedures used referring for the irradiation set-up A and B, respectively

A . 0.06 ml of CA-loaded or empty liposomes in 0.5 ml final volume of Tris-saline (pH 7.55) are placed in a silica cuvette (3 cm3 ) positioned, at five minutes intervals, in the TDS and starting the irradiation at room temperature (23°C). . At the end of exposures (60 min), one cuvette is withdrawn, at five-minute intervals, and immediately 2.4 ml Tris-saline (pH 7.55) and 0.1 ml of substrate p-nitrophenyl acetate (2 mM final concentration) are added. THz-BRIDGE progress report February 1 to July 31, 2002 21

. The kinetic measurement is immediately made, following for 2-3 minutes the appearance of reaction product p-nitrophenolate anion at its peak absorbance (=400 nm) on a Cary 50 Scan spectrophotometer. . Exposure of sham samples is performed in the same manner with none THz radiation . Aliquots of preparations of CA-loaded or empty liposomes, maintained at their optimal storage temperature of 4°C for 60 min, are removed and assayed for the enzyme activity in order to evaluate the overall liposome preparations’ stability.

. 0.06 ml of CA-loaded or empty liposomes in 2.84 ml final volume of Tris-saline (pH 7.55) are placed in a silica cuvette (3 cm3 ), positioned in the modified cell-beam of spectrophotometer (Fig. 17). . The THz radiation is started and after 30” 0.1 ml of substrate p-nitrophenyl acetate (2 mM final concentration) added. . The kinetic measurement is made during the THZ irradiation, following for 2-3 minutes the appearance of reaction product p-nitrophenolate anion at its peak absorbance (=400 nm) on an Cary 50 Scan spectrophotometer. . Exposure of sham samples is performed in the same manner with none THz radiation.

In both sets of exposure, the kinetic measurements are also carried out separately with empty- liposomes to measure the contribution due both to light scattering of the liposome suspensions and to self-hydrolysis of the substrate.

Enzyme activity measurements

The substrate p-nitrophenyl acetate hydrolysis rate, expressed as change in absorbance units at 400 nm/min (A/min), is computed automatically as the slope of a linear fit to experimental recorded curves (Fig. 18). Data from exposed and sham samples are compared and subjected to an analysis of variance (ANOVA). If the F value is significant the unpaired two-tailed Student’s t-test is used to compare the exposed and sham samples at 99% confidence interval.

Results

The results of the experimental activities are reported in Table 5 and Fig. 18, where the substrate influx rate (measured as A/min) is reported as a function of the pulse repetition rate and at different values of the average power delivered to the sample. As one can see from the Table, in both setup A and B there is a difference of p-PNA hydrolysis rate between exposed and control samples. The difference of p-PNA hydrolysis rate is significantly higher in setup B, since the real time observations reduce the presence of possible reversible effects. More specifically a highly significant increase of the substrate influx rate is present at the repetition rate of 7 Hz, both in setup A and B. In spite of an intrinsic variability of the membrane system due to the use of different liposome preparations throughout all the experiments, the observed effects cannot be merely due to a difference of average power delivered to the system. In conclusion, our preliminary results indicate that the membrane model used in these studies appears to be a system sensitive to THz exposure. Resonance effects on cationic liposomes entrapping carbonic anhydrase have already been observed in the ELF (Extremely low frequencies) THz-BRIDGE progress report February 1 to July 31, 2002 22 region, at 7 Hz. Future studies will provide further information about any possible correlation between resonance effects observed at a pulse repetition rate of 7 Hz and the effects observed in the ELF region.

Room Time of Power Pulse A/min a/min %CA %CA Enzyme Setup Temp. Exposure (mW/cm2 ) repetition Control Exposed Enzyme activity (°C) rate activity exposed control A 20-22 60’ 0.165 4sec/5 Hz 0.0064  0.0015 0.0059  0.0022 34  8 28  9 (n=18) (n=20)

A 20-22 60’ 0.231 4sec/7 Hz 0.0024  0.0018 0.0036  0.0019 16  12 24  13 (n=8) (n=10) (p=0.100)

B 26-29 2’ 5.6 4sec/5 Hz 0.0025  0.0011 0.0027  0.0018 21  10 (n=18) (n=25) 23  14

B 22 2’ 7.8 4sec/7 Hz 0.0027  0.0014 0.0058  0.0014 14 8 31  9 (n=22) (n=27) (p=8,6E- 10)

B 29 2’ 11.1 4sec/10 Hz 0.0034  0.0010 0.0046  0.0014 28  8 37  12 (n=7) (n=9) (p=0.041)

Table 5. Results of the irradiation experiments at ENEA

THz effects on CA-loaded Liposomes

Sham 50 Exposed

40 y t i v i t c

c i t a m

y 20 z n E

0 5B 7B 10B 7A Frequency Pulsed (Hz)

Figure 18: Enzimatic activity at different frequencies and with different setup. THz-BRIDGE progress report February 1 to July 31, 2002 23

WP-3 : Safety issues at specific occupational sites

The questionnaire has been further distributed to various labs and put in an interactive form on the project web-page by ENEA. Only two forms have been returned in this progress period adding to the 16 groups that had already returned completed questionnaire providing information about 27 different THz systems. THz-BRIDGE progress report February 1 to July 31, 2002 24

Appointment of personnel Dr David Newnham was appointed to the Spectroscopy Group at TeraView Limited in July 2003. Mr Thomas Lo was also appointed to the Spectroscopy Group at TeraView Limited in June 2003 for a period of four months.

Conference papers

Invited talk at the Royal Society Scientific Discussion Meeting on “The terahertz gap: the generation of far- infrared radiation and its applications”, P.F. Taday, “Applications of terahertz spectroscopy to medical and pharmaceutical science”, June 2003.

Invited talk at 2nd, International Conference on Biomedical Spectroscopy, London. P.F. Taday, July 2003

Published papers

“Damaging DNA bases by light over a wide spectral range” E. Sarantopoulou, Z. Kollia, A. C. Cefalas, S. Kobe. - Jour. Biol. Phys. 29(2-3) 149-158 (2003).

P.F. Taday, I.V. Bradley, D.D. Arnone, “Terahertz Pulse Spectroscopy of Biological Materials: L-Glutamic Acid”, Journal of Biological Physics, 29(2-3) 109-115 (2003).

Ruth M.Woodward, Vincent P.Wallace, Richard J. Pye, Bryan E. Cole, Donald D. Arnone, Edmund H. Linfield, and Michael Pepper “Terahertz Pulse Imaging of ex vivo Basal Cell Carcinoma”, Journal of Investigative Dermatology, 120(1) ,72-8 (2003). THz-BRIDGE progress report February 1 to July 31, 2002 25

Papers presented by the project partners at the THz-BRIDGE Workshop (Proceedings will be published as a special issue of Journal of Bological Phyosics)

Restrahlen to T-Rays – 100 Years of Terahertz Radiation M.F. Kimmitt Journal of Biological Physics, 29 (2003), 77-85

Trends and Perspectives in Molecular Spectroscopy: An Introduction W. Mäntele Journal of Biological Physics, 29 (2003), 87-88

THz-Spectroscopy of Biological Molecules T.R. Globus, D.L. Woolard, T. Khromova, T.W. Crowe, M. Bykhovskaia, B.L. Gelmont, J. Hesler, A.C. Samuels Journal of Biological Physics, 29 (2003), 89-100

Experimental Determination of the Far-Infrared Optical Properties of Biological Matter in Aqueous Solution A. Matei, M. Dressel Journal of Biological Physics, 29 (2003), 101-108

Terahertz Pulse Spectroscopy of Biological Materials: L-Glutamic Acid P.F. Taday, I.V. Bradley, D.D. Arnone Journal of Biological Physics, 29 (2003), 109-115

Terahertz Time-Domain Spectroscopy of Glucose and Uric Acid P.C. Upadhya, Y.C. Shen, A.G. Davies, E.H. Linfield Journal of Biological Physics, 29 (2003), 117-121

Catalogue of Human Tissue Optical Properties at Terahertz Frequencies A.J. Fitzgerald, E. Berry, N.N. Zinov'ev, S. Homer-Vanniasinkam, R.E. Miles, J.M. Chamberlain, M.A. Smith Journal of Biological Physics, 29 (2003), 123-128

Determination of Glucose Concentration in Whole Blood using Fourier-Transform Infrared Spectroscopy Y.C. Shen, A.G. Davies, E.H. Linfield, P.F. Taday, D.D. Arnone, T.S. Elsey Journal of Biological Physics, 29 (2003), 129-133

Light-induced Difference Terahertz Spectroscopy Y.C. Shen, P.C. Upadhya, A.G. Davies, E.H. Linfield Journal of Biological Physics, 29 (2003), 135-139

Two Methods for Modelling the Propagation of Terahertz Radiation in a Layered Structure G.C. Walker, E. Berry, S.W. Smye, N.N. Zinov'ev, A.J. Fitzgerald, R.E. Miles, M. Chamberlain, M.A. Smith Journal of Biological Physics, 29 (2003), 141-148

Photodissociation Dynamics of DNA Bases E. Sarantopoulou, Z. Kollia, A.C. Cefalas, S. Kobe, Z. SamardŽija Journal of Biological Physics, 29 (2003), 149-158

Absorption and Diffusion Measurements of Biological Samples using a THz Free Electron Laser E. Giovenale, M. D'Arienzo, A. Doria, G.P. Gallerano, A. Lai, G. Messina, D. Piccinelli Journal of Biological Physics, 29 (2003), 159-170

THz Exposure of Whole Blood for the Study of Biological Effects on Human Lymphocytes M.R. ScarfÌ, M. RomanÒ, R. Di Pietro, O. Zeni, A. Doria, G.P. Gallerano, E. Giovenale, G. Messina, A. Lai, G. Campurra, D. Coniglio, M. D'Arienzo Journal of Biological Physics, 29 (2003), 171-176

Effects of THz Exposure on Human Primary Keratinocyte Differentiation and Viability R.H. Clothier, N. Bourne Journal of Biological Physics, 29 (2003), 179-185

On-Chip THz Detection of Biomaterials: A Numerical Study T. Baras, T. Kleine-Ostmann, M. Koch Journal of Biological Physics, 29 (2003), 187-194

Status of THz-to-Visible Nanospectroscopy Development F. Keilmann Journal of Biological Physics, 29 (2003), 195-199

Biological and Biomedical Applications of Synchrotron Infrared Microspectroscopy P. Dumas, L. Miller Journal of Biological Physics, 29 (2003), 201-218

Synchrotron-based Biological Microspectroscopy: From the Mid-Infrared through the Far-Infrared Regimes L.M. Miller, G.D. Smith, G.L. Carr Journal of Biological Physics, 29 (2003), 219-230

A Near Field Microscope in the Tera-hertz Region A. Doria, A. Coniglio Journal of Biological Physics, 29 (2003), 231-237

Measurement of THz Spot Sizes with a ?/200 Diameter in the Near-Field of a Metal Tip N.C.J. van der Valk, P.C.M. Planken Journal of Biological Physics, 29 (2003), 239-245

T-ray Imaging and Tomography S. Wang, B. Ferguson, D. Abbott, X.-C. Zhang Journal of Biological Physics, 29 (2003), 247-256

Terahertz Pulsed Imaging of Skin Cancer in the Time and Frequency Domain R.M. Woodward, V.P. Wallace, D.D. Arnone, E.H. Linfield, M. Pepper Journal of Biological Physics, 29 (2003), 257-259

Risk Perception and Safety Issues E. Berry Journal of Biological Physics, 29 (2003), 263-267

Perception of Risks from Electromagnetic Fields: Lessons for the Future P. Vecchia Journal of Biological Physics, 29 (2003), 269-274

Synchrotron-Based FTIR Spectromicroscopy: Cytotoxicity and Heating Considerations H.-Y.N. Holman, M.C. Martin, W.R. McKinney Journal of Biological Physics, 29 (2003), 275-286

Generation of Narrowband Tunable THz-Radiation via Optical Rectification in Periodically Poled Materials G. Torosyan, K. Nerkararyan, Y. Avetisyan, R. Beigang Journal of Biological Physics, 29 (2003), 287-293

Performance Characteristics of a Smith-Purcell Tunable Terahertz Source M. Mross, T.H. Lowell, R. Durant, M.F. Kimmitt Journal of Biological Physics, 29 (2003), 295-302

Properties and Planned Use of the Intense THz Radiation from ELBE at Dresden-Rossendorf K. Fahmy, G. Furlinski, P. Gippner, E. Grosse, H. Piest, W. Seidel, M. Sczepan, D. Wohlfarth, A. Wolf, R. Wuensch Journal of Biological Physics, 29 (2003), 303-307

The Synchrotron Infrared Activities at BESSY U. Schade, W.B. Peatman Journal of Biological Physics, 29 (2003), 309-312

Terahertz Radiation at ANKA, the New Synchrotron Light Source in Karlsruhe Y.-L. Mathis, B. Gasharova, D. Moss Journal of Biological Physics, 29 (2003), 313-318

Very High Power THz Radiation Sources G.L. Carr, M.C. Martin, W.R. McKinney, K. Jordan, G.R. Neil, G.P. Williams Journal of Biological Physics, 29 (2003), 319-325

Tunneling Processes Induced by Terahertz Electric Fields S.D. Ganichev, I.N. Yassievich, W. Prettl Journal of Biological Physics, 29 (2003), 327-334

Optical Constants of Two Typical Liquid Crystals 5CB and PCH5 in the THz Frequency Range R.-P. Pan, T.-R. Tsai, C.-Y. Chen, C.-L. Pan Journal of Biological Physics, 29 (2003), 335-338

Publications and other conference presentations in 2002

S. Mair, B. Gompf, M. Dressel: ``Microspectroscopy and Imaging in the THz Range Using Coherent CW Radiation'' to be published in Physics in Medicine and Biology, November 2002 (physics/0202039)

M. Dressel: ``Near-Field Spectroscopy'' paper presented at the Workshop “Forum Photonics BW: Moderne Optik, aktuelle Trends aus der Grundlagenforschung”, Stuttgart, Juli 2002

G.P. Gallerano, “The THz-BRIDGE project” Paper presented at the COST281 Meeting, Rome, May 2-5 , 2002

E. Giovenale, “The THz-BRIDGE project” Paper presented at the 3rd GR-I Conference, Patrass (Greeve), September5-10, 2002