1 BIOFYSIKKMØTET 2004 KONGSVOLD FJELDSTUE, MARCH 17th-19th, 2004 PROGRAM AND ABSTRACTS - Post-Meeting Version, Faggruppe for Biofysikk Norsk Fysisk Selskap
2 PREFACE The board of Faggruppe for Biofysikk in Norsk Fysisk Selskap wishes you welcome to Biofysikkmøtet In accordance with a nice tradition the three-day meeting is also this year held at Kongsvold Fjeldstue between 17th and 19th of March. The attached abstracts demonstrate a significant and wide-ranged activity in Norwegian biophysics. The Kongsvold meetings also contribute to maintain and strengthen the contacts between the various biophysics groups in Norway and provide a forum where students, stipendiates, teachers and researchers can meet and interact in pleasant surroundings. In addition, the invited talks (many from foreign guests) introduce our community to new research topics. The board wants to thank Norsk Fysisk Selskap and Norges landbrukshøgskole for economical and other support. You are all welcome to Biofysikkmøtet 2004! For the board of Faggruppe for Biofysikk, Norsk Fysisk Selskap Gaute T. Einevoll /leader/
3 BIOFYSIKKMØTET 2004 KONGSVOLD FJELDSTUE, MARCH 17th 19th 2004 CONTENT Preface Meeting program Posters List of participants Abstracts
4 MEETING PROGRAM Wednesday, March 17th 10:32 Arrival of train from Trondheim at Kongsvold 12:23 Arrival of train from Oslo at Hjerkinn 10:00-14:30 Registration finding your room 13:30-14:30 LUNCH 14:30-14:35 Opening of meeting, Gaute T. Einevoll, NLH 14:35-15:20 IT-1: Ralf Metzler, NORDITA: Bubbles in DNA: Equilibrium fluctuations and dynamics 15:20-16:05 IT-2: Anders Johnsson, NTNU: Experimental and theoretical studies of oscillations in the transpiration of plants 16:05-16:30 COFFEE BREAK 16:30-16:55 O-1: Ane V. Vollsnes, Ane Jerstad, Cecilia Futsæther, Unni Oxaal and Thor Bernt Melø, NLH & NTNU: Plant root morphology responses to ozone or illumination 16:55-17:20 O-2: Einar Sagstuen, Eli O. Hole, Randi Vågane, David M. Close and William H. Nelson, UiO, ETSU, GSU: Charge transfer in peptide nucleic acid base complexes: An EPR, ENDOR and DFT study 17:20-17:45 O-3: André Krivokapic, Eirik Malinen og Einar Sagstuen, UiO: Langtrekkende strålingsindusert hulltransport i et donor-akseptorsystem bestående av krystallinsk cytosin dopet med tiocytosin 17:45-18:10 O-4: Eirik Malinen, Tor Arne Vestad, Elin Agathe Hult, Eli Olaug Hole og Einar Sagstuen, UiO & SSV: Differensiell fotonabsorpsjon i faste stoffer - problemstillinger og anvendelser 18:10-18:30 COFFEE BREAK 18:30-19:15 IT-3: Johan Moan, DNR & UiO: Festforedrag: Light the most important physical factor in biology 19:30 DINNER Thursday, March 18th 07:30-08:30 BREAKFAST 08:45-09:30 IT-4: Simon R. Schultz, UCL: Two-photon imaging of neural activity in the intact brain 09:30-09:55 O-5: Klas Pettersen and Gaute T. Einevoll, NLH: From extracellular electric potentials to neural activity: How can we interpret laminar-electrode recordings from cortex? 09:55-10:20 O-6: Gaute T. Einevoll, Martin Kermit and Anders M. Dale, NLH & Harvard- MGH: Combined statistical and mechanistic modeling of cortical population responses measured with laminar electrodes 10:20-10:35 COFFEE BREAK 10:35-11:00 O-7: Hans E. Plesser and Gaute T. Einevoll, NLH: COTHACO: A comprehensive model of the thalamocortical pathway 11:00-11:30 POSTER ADVERTISEMENTS 11:30-14:00 RECREATION (F.EX. SKIING) 14:00-15:00 LUNCH 15:00-16:15 POSTER SESSION 1: P1-P7 16:15-16:45 COFFEE BREAK
5 16:45-18:00 POSTER SESSION 2: P8-P14 18:00 ELECTION AND DISCUSSION - Election of new board - Discussion on future developments of Norwegian biophysics - Discussion on possible role of Faggruppe for biofysikk in Year of Physics, :30 DINNER Friday, March 18th 07:30-08:30 BREAKFAST 08:45-09:30 IT-5: Mikael Lindgren, Mikael Tiihonen, Fredrik Laurell, Per Hammarström, NTNU, KTH & LiTH: A novel UV laser source for fluorescence excitation of proteins 09:30-09:55 O-8: Ingunn Tufto, David Byberg, Kirsten H. H. Nygaard, Catharina de Lange Davies, NTNU: Transient perfusion in tumours treated with collagenase or hyaluronidase 09:55-10:15 COFFEE BREAK 10:15-10:40 O-9: Signe Danielsen, Marit Sletmoen, Gjertrud Maurstad and Bjørn T. Stokke, NTNU: Biopolymer assembly and interactions: Nanoscale studies using AFM 10:40-11:05 O-10: Lars Eric Sæther, Berit Falch, Dag Roar Hjelme, Bjørn Torger Stokke, NTNU & Optomed: Polymer gel signal transducers as interface for optical detection of biological macromolecules in vitro and in vivo 11:05 Closing of meeting 11:15-12:30 LUNCH ~ 11:45 Departure to Hjerkinn; train to Trondheim leaving Hjerkinn at ~ Departure to Hjerkinn; train to Oslo leaving Hjerkinn at [Note: Later trains to Oslo and Trondheim are also possible; some even stop at Kongsvoll.]
6 POSTER SESSION P-1: Hilde Nortvedt Andersen, Arne Erikson, and Catharina de Lange Davies, NTNU: The impact of the extracellular matrix structure on the uptake of therapeutic macromolecules in solid tumours P-2: Ingunn Bjørhovde og Erik O. Pettersen, UiO: Cellers respons på bestråling med β-partiklar frå inkorporert [ 3 H]-valin ved ultra-lave doseratar P-3: Patrick Blomquist, John Wyller, and Gaute T. Einevoll, NLH: Population models for neural activity in cortex P-4: Miguel Cavero, Klas H. Pettersen and Gaute T. Einevoll, NLH & UKN: From extracellular electric potentials to neural activity: Signature of action potentials for realistic neuron populations P-5: Kanutte Christiansen og Erik O. Pettersen, UiO: Adaptiv effekt av små akutte røntgenstråledoser for celler dyrket i kultur P-6: Robert Hansen, Hanne Martinusen, Live Eikenes and Christian Brekken, NTNU & St.Olav Hospital: The effect of collagenase on vascular volume and permeability in tumors P-7: Linda Helander og Anders Johnsson, NTNU: Dose-respons for PDT-behandling og osmose effekter på Jurkat P-8: Ellen Marie Husby, Liv Marit Rønning, Live Eikenes, and Catharina de Lange Davies, NTNU: The effect of collagenase and hyaluronidase on diffusion of macromolecules in multicellular spheroids P-9: Ane Jerstad, Ane V. Vollsnes, Aud B. Eriksen, Unni Oxaal and Cecilia M. Futsaether, NLH: Does ozone affect the root growth of plants? P-10: Martin Kulhawczuk og Anders Johnsson, NTNU: Produksjon av porfyriner i P.acnes ved inkubering med 5-ALA og Metyl 5-ALA P-11: Morten Kr. Off, Pavel Vorobey, Alexander Vorobey and Johan Moan, NTNU, DNR & Minsk: Spectral investigation of folic acid photodestruction in different solutions P-12: Ståle Ramstad and Anders Johnsson, NTNU: A study of ALA and ALA ester incubated P. acnes bacteria P-13: Tor A. Vestad, Eirik Malinen, Anders Lund, Eli O. Hole and Einar Sagstuen, UiO & Linköping Univ.: Properties of Formates for EPR Dosimetry Purposes P-14: Inger Camilla Walle, Taran Paulsen Hellebust og Dag Rune Olsen, DNR &UiO: Stråledose til ovariene ved høyenergetisk strålebehandling
7 LIST OF PARTICIPANTS Hilde Nortvedt Andersen NTNU Ingunn Bjørhovde UiO Patrick Blomquist NLH Miguel Cavero NLH Kanutte Christiansen UiO Signe Danielsen NTNU Catharina Davies NTNU Gaute Einevoll NLH Robert Hansen NTNU Linda Helander NTNU Eli Olaug Hole UiO Ellen Marie Husby NTNU Ane Jerstad NLH Anders Johnsson NTNU Andre Krivokapic UiO Martin Kulhawczuk NTNU Mikael Lindgren NTNU Eirik Malinen UiO Ralf Metzler NORDITA Johan Moan DNR & UiO Morten Off NTNU & DNR Unni Oxaal NLH Klas Pettersen NLH Hans Ekkehard Plesser NLH Ståle Ramstad NTNU Einar Sagstuen UiO Simon Schultz UC London Kristin Sæterbø NTNU Lars Eric Sæther NTNU Ingunn Tufto NTNU Ane V. Vollsnes NLH Inger Camilla Walle UiO & DNR
8 Bubbles in DNA: Equilibrium fluctuations and dynamics IT-1 Ralf Metzler NORDITA NORDIC INSTITUTE FOR THEORETICAL PHYSICS BLEGDAMSVEJ 17, DK-2100 KØBENHAVN Ø Even at physiological temperatures, double-stranded (ds) DNA incessantly opens up floppy single-stranded (ss) bubbles due to thermal fluctuations. Due to the comparatively strong stacking (DNA base pair-base pair) interactions in dsdna which supplement the Watson- Crick bonds within a base pair, this process of bubble formation is co-operative, i.e., once a bubble opens, a whole stretch of dsdna opens up at a time. At physiological temperatures, the typical length of bubbles is 20 to 30 base pairs. These bubbles constantly open (unzip) and close (zip) as a random process, which was recently monitored on the single molecular level by molecular beacons. The fluctuation can theoretically be described in terms of a Fokker- Planck equation for the bubble size, and characteristic life-times of bubbles can be obtained through a first passage time analysis. It turns out that the expression obtained from the free energy of the classical Poland-Scheraga model of DNA nicely reproduces the experimental results, but also gives rise to a correction of the deduced (un)zipping rates. The DNA bubbles are, inter alia, important for ss-binding proteins, which can attach to the DNA if they catch such an open bubble. It has been identified that there is a kinetic barrier, stemming from a competition between the life-time of a bubble and the characteristic binding time of these proteins. By modifying such an ss-binding protein, one can vary its binding rates, and thus study this competition of time scales. At increased temperatures, the dsdna finally melts completely, i.e., it denatures fully into two double-strands. From the experimental melting data, it appears that the melting transition is rather sharp. It has been argued that it actually might be a first order phase transition. There is currently an ongoing debate how one can obtain such a first order transition from theoretical models. Finally, it is of current interest how the DNA bubbles and DNA binding proteins actually move (i.e., diffuse) along the dsdna. This has been studied in terms of the random energy model, revealing subdiffusion. I will give a short overview of recent experiments and summarise recent approaches. Further reading:  D Poland and HA Scheraga, Theory of Helix-Coil Transitions in Biopolymers (Academic, New York, 1970)  A Hanke and R Metzler, J Phys A 36 (2003) L473  Y Kafri et al, Phys Rev Lett 85 (2000) 4988; ibid 90 (2003) ; A Hanke and R Metzler, ibid 90 (2003)  G Altan-Bonnet et al, Phys Rev Lett 90 (2003)  K Pant et a;. J Mol Biol 327 (2003) 571  T Hwa et al, Proc Natl Acad Sci USA 100 (2003) 4411  M Slutsky et al, eprint q-bio.bm/
9 Experimental and theoretical studies of oscillations in the transpiration of plants IT-2 Anders Johnsson DEPARTMENT OF PHYSICS NTNU, N-7491 TRONDHEIM The water status of plants is of great interest in a global context as well as from a scientific point of view. The transpiration of plants represents an overall loss of water into the atmosphere, estimated to about 70% of water taken up by the roots. Studies of plant water regulation and possible means to control it is, therefore, of importance.. A short overview of the main parameters in the general water transport system of plants will be given. The biophysical modelling of the water transport must take the differents pathways of water thought the plant into consideration. The stomata openings of the leaves (or other parts of plants) control the gas exchange: transpiration of water vapour and simultaneous carbon dioxide uptake to the photosynthesic apparatus. Of central importance in this regulation is the guard cells of the stomatal appartus. The water pathways also involve the water entrance into and out of cells and thus the water permeabilities of cell membranes. In this context a short review of the aquaporins in plants membranes will be given. A modelling of the plant water control system requires at least three dynamic elements and must, therefore, be modelled as at least a three-variable, non-linear system. One characteristics of such non-linear systems is the possibility for oscillations. A presentation of oscillatory transpiration control in plants will be given as well as results on the cellular level. Finally, experimental results showing a high variety of complex oscillatory patterns, possible to show up in such multivariable systems will be discussed. They may represent a possible route to a chaotic system. The modelling of the water transport system will be discussed.
10 Light the most important physical factor in biology IT-3 Johan Moan INST: FOR CANCER RESEARCH; DNR; MONTEBELLO; OSLO 3, AND DEPT.OF PHYSICS, UIO, OSLO, NORWAY Life has literally developed in radiation from the sun. Ultraviolet radiation (UV) has been a driving force in the evolution through its generation of mutations and through its killing of sensitive organisms. Practically all energy on Earth is provided by the sun; the major pathway to useful energy being photosynthesis. Our main perception of the world is collected by vision. Our visual images constitute the framework of our construction of the universe in our mind. In such a short presentation of photobiophysics there is time for paying attention to only a few aspects of our topic. I have chosen circannual and circadian rhythms and sun-induction of vitamin D in our skin. Circannual rhythms have essentially two components: one related to- and adjusted by the ratio of the length of the day and that of the night, and one inherent in the body and unrelated to light. A number of parameters in the physiology and behaviour of animals show regular annual rhythms: levels of sexual hormones, hibernation, migration to other latitudes, loss of horns, depression, seasonal affective disorders (SAD), suicide rates, birth rates, and even initiation of wars. A high level of testosterone of young men appears to be among the driving forces of starting fighting and wars. Circadian rhythms have a period of about 24 hours. In humans the length of the free-running period is on the average hours, and is adjusted to 24 hours by daylight. In almost all cells of the body there are circadian clocks. A large number of genes are periodically active. The master clock is believed to reside in two small glands in the brain: the suprachiasmatic nuclei, SCN, which receive signals directly from specialized detectors in the retina ( not from cones and rods). From SCN signals are sent via the upper part of the spinal chord to the pineal organ in the brain. This gland produces the night hormone, melatonin, when it is dark. Melatonin plays a major role in the conduction of a large division of the hormone orchestra. The impact of the circadian rhythms in our lives is large: we are coldest at 4.30, the blood pressure increases fastest at 07.00, we are most awake at 10.00, our coordination of movements is optimal at 14.00, our heartbeats are strongest and our muscular strength is largest at 17.00, the blood pressure is maximal at 18.00, we are warmest at 19.00, melatonin is produced from 21.00, the peristaltic movements cease at 23.00, and our sleep is deepest at SAD seems to be related to irregularities of the circadian machinery. Solar radiation is an important source of vitamin D. This vitamin is a prohormone, calcitriol being the active vitamin D- derived hormone. The level of another vitamin D derivative, calcidiol, is % larger in summer than in winter. Vitamin D seems to protect against cancer, or at least, slow down its progression. We have recently shown that the prognosis of several forms of cancer is dependent on the season when the treatment is started. The season of best prognosis coincides with that when the status of vitamin D is optimal: late summer. Thus, it is likely that UV from the sun can both induce cancer and protect against it. This dilemma should be presented to the public in a constructive way.
11 Two-photon imaging of neural activity in the intact brain IT-4 Simon R. Schultz WOLFSON INSTITUTE FOR BIOMEDICAL RESEARCH AND GATSBY COMPUTATIONAL NEUROSCIENCE UNIT, UNIVERSITY COLLEGE LONDON, UK Our everyday sensory experience arises from the co-activation of large ensembles of neurons during behaviour and in response to stimulation from the world around us. To study the neural basis of sensory information processing, previous approaches have focused on recording the electrical activity of a small number (usually one) of cells randomly sampled by the tip of a microelectrode - or, at the other extreme, of haemodynamic responses reflecting the average activity in a volume of tissue, to which the contribution of individual neurons is not resolved. Multi-photon fluorescence microscopy  offers a new experimental approach to the study of sensory coding, in which physiological events such as calcium transients can be studied at sub-cellular spatial resolution, and fast (millisecond scan-line) temporal resolution. Twophoton excitation offers several advantages over single-photon microscopy which make it viable as a systems neuroscience tool, including greater depth penetration due to lower scattering, meaning it can be used in vivo, and confinement of the fluorescence signal to the vicinity of the focal point, i.e. specificity. We are currently pursuing several applications of two-photon microscopy to the study of neuronal circuitry in the rodent cortex and cerebellum. The first is the patch and fill experiment, in individual neurons are filled with calcium indicator dye through a patch-clamp seal, and calcium transients in dendrites evoked by tactile or visual stimuli. The second type of experiment is bulk-loading, where a membrane-permeable dye is pressure-ejected into the brain at a depth of several hundred microns . The dye is taken up by neurons, enabling hundreds of cells in the locality to be visualised simultaneously. A third approach which may prove useful for analysing the cortical circuit is targeted recording, in which specific cell types (such as GABA-ergic interneurons) are labelled. They can then be visualised under the microscope, and targeted for extra- or intra-cellular recording.  W. Denk et al, Science 248:73-76 (1990)  C. Stosiek et al, Proc. Nat. Acad. Sci. USA 100(12): (2003).
12 A novel UV laser source for fluorescence excitation of proteins IT-5 Mikael Lindgren Department of Physics, NTNU 7491 Trondheim, Norway Mikael Tiihonen, Fredrik Laurell Department of Physics, KTH Stockholm, Sweden Per Hammarström Department of Biochemistry, LiTH Linköping, Sweden Modern photonics technology enables new means to study biological compounds by means of fluorescence imaging and spectroscopy. A vast number of biological compounds absorb only in the UV and particularly the wavelength region for protein excitation is difficult to reach with one-photon excitation using conventional laser sources. Here we report on a compact parametric oscillator (OPO) with intracavity sum-frequency generation (SFG) to generate 293 nm UV laser irradiation. The OPO/SFG device was pumped by a 100 Hz Nd:YAG laser (1064 nm) of own design, including subsequent second harmonic generation (SHG) in an external periodically poled KTiOPO4 (KTP) crystal. The whole system could be used to deliver more than 30 µj laser irradiation per pulse 100 Hz). It is shown how aqueous samples ovalbumin or bacteria spores (Bacillus subtilis) can be excited by the UV-light at 293 nm resulting in strong fluorescence emission in the range nm. We also give examples on time-resolved fluorescence energy transfer between tryptophans and fluorophores attached to protein structures (TTR), and how it can be used to monitor the dynamical state of aggregated proteins.
13 Plant root morphology responses to ozone or illumination O-1 Ane V. Vollsnes, Ane Jerstad, Cecilia Futsæther, Unni Oxaal and Thor Bernt Melø * DEPARTMENT OF MATHEMATICAL SCIENCES AND TECHNOLOGY AGRICULTURAL UNIVERSITY OF NORWAY, ÅS; * DEPARTMENT OF PHYSICS, THE NORWEGIAN UNIVERSITY OF SCIENCE AND TECHNOLOGY, TRONDHEIM By growing plants in transparent agarose gel, the roots are accessible to observation during an experiment . This fact has been taken advantage of in a study where clover plants (Trifolium subterraneum, a particularly ozone sensitive species ), were subjected to ozone treatments . Plant roots were photographed daily and the pictures analysed to find a measure of their size at each point in time. Thereby the variation in root growth rates could be studied. The number of secondary roots and the timing of their emergence were also recorded. Some results from these studies will be presented. To photograph the plant roots in the study described above, the roots had to be illuminated. Electroluminescent sheets giving a light intensity of about 0.5 µmol m -2 s -1 in the PAR (photosynthetically active radiation) region were used. In a different study the possible effect of this illumination on lentil (Lens culinaris) roots was examined. The effects of differently coloured sheets on the amount of two pigments and on the morphology of the root systems were studied. Illumination treatments induced the formation of the two pigments differently, and the morphology of roots was also affected by illumination. A possible causal link was found between the concentration of one of the pigments and morphological traits.  C. M. Futsaether, U. Oxaal, Plant Soil (2002)  G. Pihl Karlsson et al., New Phytol (1995)  A. Jerstad et al., Poster abstract in this issue.
14 Charge Transfer in Peptide Nucleic Acid Base Complexes: An EPR, ENDOR and DFT Study O-2 Einar Sagstuen 1, Eli O. Hole 1, Randi Vågane 1, David M. Close 2 and William H. Nelson 3 1 Department of Physics, University of Oslo; 2 Department of Physics, East Tennessee State University, Johnson City, TN; 3 Department of Physics and Astronomy, Georgia State University, Atlanta, GA. Interactions between DNA and proteins are important during many stages of the cell cycle. DNA is wrapped around the histone molecules into nucleosomes which in turn are lumped into chromatin for a major part of the cycle time. Various protein-dna recognition patterns are furthermore essential for many vital processes in the cell, for instance the transcription and translation processes. It is widely recognized that protein-dna interactions are mediated by specific hydrogen bonding between various parts of different amino acids and the DNA constituents, both the bases and the sugar-phosphate backbone. Ionizing radiation ejects electrons from the given molecular system leaving holes as the primary oxidation product. This is a random distribution process. The ejected electrons eventually become captured by other molecules. These trapped electrons represent the primary reduction products from the radiation action. Most studies of radiation damages to DNA have focused on the primary oxidation and reduction products and their subsequent reactions within DNA itself. However, having amino acids closely associated with the DNA molecule, possible modifications of the radiation damage schemes worked out for isolated DNA should be considered. In a first step of doing so, specific model systems consisting of nucleic acid bases hydrogen bonded with dipeptides have been prepared in single crystal form. The radiation response of these systems has been investigated using electron paramagnetic resonance (EPR) spectroscopy and related techniques. Irradiation and measurements have been done at temperatures between 10 K and room temperature. In the present work, single crystals of the complex H H 9 N-formylglycine cytosine (see Structure at left) have been C N 5 investigated. After irradiation at 10 K, only cytosine 8 O 3 C 5 7 O anions and N-formylglycine oxidation products were C H H 4 H H N observed. Thus, a complete separation of charges seems to O 4 have taken place in the system. All reduction takes place H H 4 N at the cytosine base exhibiting the largest electron affinity All oxidation is transferred to the peptide which exhibits 1 O N H the lowest ionization potential of the two constituents. 2 Upon warming from 10 K, further evolution of the H primary radicals formed takes place, however confined to the component where the initial damage was trapped. The results obtained from the experimental studies have been complemented with theoretical calculations using Density Function Theory (DFT) methods providing further insight into the charge transfer processes involved.
15 Langtrekkende strålingsindusert hulltransport i et donorakseptorsystem bestående av krystallinsk cytosin dopet med tiocytosin André Krivokapic, Eirik Malinen og Einar Sagstuen Fysisk institutt, Universitetet i Oslo, Postboks 1048 Blindern, 0316 Oslo. Den direkte effekten av ioniserende stråling på DNA-molekylet resulterer i dannelse av frie radikaler som initielt er stokastisk fordelt mellom de ulike DNAbestanddelene. Det observeres imidlertid at stråleskadene stabiliseres selektivt; oksidasjonsproduktene lokaliseres til guanin, mens reduksjonsproduktene hovedsakelig ender opp på cytosin. Det antas at denne selektive fordelingen skyldes en transport av elektroner og hull til de energetisk mest stabile posisjonene. Etter ionisasjon vil hull således migrere til og stabiliseres i guanin, som har lavest ionisasjonspotensial, og elektroner vil migrere til og stabiliseres i cytosin som har høyest elektronaffinitet. Ionisert DNA kan derfor betraktes som et system av donorer og akseptorer som er separert av en barriere bestående av de mellomliggende baser. Til tross for omfattende forskning på dette området det siste tiåret er mekanismene for ladningstransporten i DNA og liknende systemer ennå ikke fullt ut forstått. To hovedmekanismer er foreslått; tunneling og hopping. Ved tunneling antas transporten å foregå i ett trinn fra donor til akseptor, mens hopping innebærer en multi-trinnstransport. Hopping er i litteraturen ofte beskrevet som en random walk eller en diffusjonsprosess og antas å være temperaturaktivert og lite avstandsavhengig, mens det motsatte er tilfelle for tunneling. Vi har benyttet EPR spektroskopi (electron paramagnetic resonance) for å studere strålingsindusert hulltransport i et modellsystem bestående av krystallinsk cytosin dopet med tiocytosin (se strukturene nedenfor). Tiocytosin har et mye lavere ionisasjons-potensial enn cytosin og forventes derfor å virke som en effektiv hull-akseptor. Dette systemet er bestrålt ved 10 K og studert ved ulike temperaturer. Etter bestråling er det hovedsakelig to prosesser som finner sted; transport av hull fra oksidert cytosin til tiocytosin og ladningsrekombinasjon av oksidasjons- og reduksjonsprodukter. Forskjellene mellom disse prosessene vil bli diskutert i lys av de to transport-mekanismene nevnt ovenfor. Resultatene som presenteres taler mot en diffusjon/random-walk betraktning av hulltransporten. Videre vil forutsetninger som må være oppfylt for at transporten skal finne sted bli diskutert; av spesiell interesse er hvorvidt hulldonoren må være i ionisk form. O-3 N NH 2 H N NH 2 H O N H H S N H H Cytosin Tiocytosin
16 Differensiell fotonabsorpsjon i faste stoffer - problemstillinger og anvendelser Eirik Malinen, Tor Arne Vestad, Elin Agathe Hult, Eli Olaug Hole og Einar Sagstuen Fysisk institutt, Universitet i Oslo; Statens strålevern, Østerås O-4 Absorpsjonen av røntgen- og γ-stråling er sterkt avhengig av både absorbatorens sammensetning og kvanteenergien til fotonene. Dette skyldes at ved lave fotonenergier (størrelsesorden kev) er fotoelektrisk effekt den dominerende vekselvirkningen, og dens relative betydning øker sterkt med absorbatorens atomnummer. Bestråling av stoffer med ulik atomær sammensetning kan derfor føre til store forskjeller i absorberte doser. Denne differensielle fotonabsorpsjonen vil imidlertid reduseres når fotonenergien øker, siden betydningen av fotoelektrisk effekt avtar med økende fotonenergi. I medisinsk dosimetri er målsetningen å bestemme absorbert dose til vann, siden vann er et lett tilgjengelig medium som også er organismens hovedbestanddel. For dosebestemmelser benyttes imidlertid ofte dosimetere (d.v.s. strålesensitive detektorer) som har en annen sammensetning enn vann. Dette innebærer at strålingsabsorpsjonen i dosimeteret ikke gir et direkte mål på dosen til vann. Videre vil forholdet mellom dosen til dosimeteret og dosen til vann forandre seg med fotonenergien på grunn av energiavhengigheten til den differensielle fotonabsorpsjonen. Dette skaper problemer når nøyaktige dosebestemmelser er ønskelige. På den annen side kan den differensielle fotonabsorpsjonen faktisk benyttes til å estimere fotonenergien. Hvis man har to dosimetermaterialer med ulik atomær sammensetning plassert i et strålefelt, vil doseforholdet mellom materialene kunne gi et relativt mål for fotonenergien. Hvis to slike stoffer blandes sammen og bestråles som ett dosimeter, kan man ved en EPR-spektroskopisk (Elektron Paramagnetisk Resonans) analyse finne den absorberte dosen i hver av de to bestanddelene i dette komposittdosimeteret og dermed beregne doseforholdet. Doseforholdet kan videre gis i et kalibreringsplott som funksjon av fotonenergien, og benyttes til bestemmelser av fotonenergi når strålefeltet er ukjent. Èn anvendelse av komposittdosimetere viser hvordan forholdet mellom spredt stråling og primærstråling forandrer seg med dypet i et plastmateriale. Bruken av LiF-dosimetere (i termoluminescens-dosimetri) for stråleterapi er ikke uproblematisk, siden LiF (litiumfluorid) har et høyere effektivt atomnummer enn vann. Dette innebærer at LiF absorberer en større andel lavenergetiske fotoner i et strålefelt enn det vann gjør, hvilket i sin tur fører til at dosimeteravlesningen må korrigeres for å kunne beregne dosen til vann. Siste del av foredraget vil vise hvordan den differensielle fotonabsorpsjonen mellom LiF og vann varierer med dypet i en absorbator, og hvilke konsekvenser dette får for dosebestemmelser ved bruk av LiF i dosimetri.
17 From extracellular electric potentials to neural activity: How can we interpret laminar-electrode recordings from cortex? Klas H. Pettersen and Gaute T. Einevoll DEPARTMENT OF MATHEMATICAL SCIENCES AND TECHNOLOGY AGRICULTURAL UNIVERSITY OF NORWAY, ÅS O-5 Neurons are connected through synapses. An action potential (AP) in the pre-synaptic neuron induces a trans-membrane current at the post-synaptic neuron. This synaptic current may excite or inhibit the post-synaptic neuron so that it increases or decreases the possibility of the post-synaptic neuron to produce an AP. In cortex there are many types of neurons, and each neuron could have more than thousand connections to other neurons. In building mathematical models of the neural circuitry, one often assumes populations of neurons to carry information, rather than individual neurons. However, such mathematical models for population activity have to be compared with experiments, and a promising technique for measuring such population activity is the use of so-called laminar electrodes . The laminar electrode is inserted perpendicular to cortex, and have several closely spaced recording ( µm) contacts throughout cortex. The difficulty then, is to interpret the extracellularly recorded potentials in terms of firing activity of the neurons in the population and the synaptic activity in the cortical circuit. The low-frequency part of the extracellular potentials (often called Local Field Potential (LFP)) is thought to be dominated by trans-membrane currents induced by synaptic activity in the dendrites (the high-frequency part is thought to be dominated by transmembrane currents due to APs). Here we consider a population of model neurons (based on a reconstructed pyramidal cortical cell ) and calculate trans-membrane currents following synaptic inputs. These trans-membrane currents are then used to calculate the LFP at positions corresponding to the electrode contact points for our model system. The commonly used procedure for translating the potentials to synaptic activity is called the current-source density method (CSD method) . This method finds the corticaldepth distribution of trans-membrane currents (current-source density) assuming homogeneous synaptic activity throughout the cortical plane. The applicability of the method to realistic cortical systems has been debated , and here we test this method for our model system (where we know the result). We further propose an alternative procedure for computing the current source density in cortex when the population activity is confined to a column (such as in the rat-whisker system).  I. Ulbert et al., J. Neurosci. Meth (2001)  Z. F. Mainen and T. J. Sejnowski, Nature (1996)  C. Nicholson and J. A. Freeman, J Neurophysiol (1975)
18 O-6 Combined statistical and mechanistic modeling of cortical population responses measured with laminar electrodes Gaute T. Einevoll, Martin Kermit and Anders M. Dale * DEPARTMENT OF MATHEMATICAL SCIENCES AND TECHNOLOGY AGRICULTURAL UNIVERSITY OF NORWAY, ÅS; * NMR CENTER, DEPARTMENT OF RADIOLOGY, HARVARD-MGH, BOSTON The new non-invasive imaging method fmri has, combined with alternative complementary techniques such as PET, EEG and MEG given a wealth of new information about the function of the brain at the systems level. However, the spatial resolution of these techniques is essentially limited to the systems levels, and it has been difficult to firmly link the observed systems-level activity with electrical or metabolic activity of the underlying neuronal circuitry. On the microscopic level invasive single-unit electrophysiological recordings have for more than half a century given significant insights into the function of single neurons. From a modeling point of view the establishment of compartmental modeling of neural electrophysiological activity now provides a relatively firm foundation for mathematical exploration of the properties of biological neural networks (see, for example, ). However, attempts of "bridging the gap" between the microscopic single-unit and the macroscopic systems level have so far had limited success. Laminar-electrode recording is a promising experimental techniques for studying neural circuits at the mesoscopic level (~0.1-1 mm). Here the extracellular potential, generated by ion currents crossing the neuron membrane, is measured at ~20 different cortical depths with a typical spacing of µm. The laminar-electrode data potentials are thought to reflect the firing of action potentials originating in the soma (high-frequency part) and dendritic processing synaptic currents (low-frequency part). The mathematical analysis of such data has until now been limited to standard statistical methods such as principal components analysis (PCA). However, the interpretation of the data in terms of firing activity of cortical neuron populations have not been possible. Here we present a new mathematical scheme for making such identifications. where we use physiological and biophysical insights to constraint the statistical modelling. Examples of application of the new mathematical method to stimulus-averaged laminar-electrode data (recorded at Harvard MGH) from the rat whisker cortex following single-whisker stimulations, will be presented.  Computational neuroscience: Realistic modeling for experimentalists, ed. E. de Schutter, CRC Press, London, 2000.
19 O-7 COTHACO: A Comprehensive Model of the Thalamocortical Pathway Hans E. Plesser and Gaute T. Einevoll DEPARTMENT OF MATHEMATICAL SCIENCES AND TECHNOLOGY AGRICULTURAL UNIVERSITY OF NORWAY, ÅS Over the past years, several studies have demonstrated that feedback plays an important role for visual processing in later stages of the visual pathway, from primary visual cortex upward; see  for a review. In the early visual pathway, the picture is less clear-cut at present. If we set aside eye movements, there is no feedback into the retina. In the dorsal lateral geniculate nucleus (dlgn), on the other hand, only some 10% of all synapses convey ascending input from the retina, while the vast majority mediates descending signals from visual cortex . Most ascending synapses are located close to the soma of LGN relay cells, though, and are very strong: an individual retino-geniculate spike will elicit a relay-cell spike with high likelihood. There is indeed strong evidence that thalamic relay cells fire only in immediate response to retinal input; figuratively speaking, relay cells can only knock spikes out of retinal spike trains, never insert new spikes . LGN relay cells thus have circular symmetric receptive fields similar to those found in the retina, albeit sharper. LGN has therefore long been considered a passive relay between retina and visual cortex. It appears implausible, though, that evolution should have retained the massive corticothalamic feedback without purpose. Several groups have indeed demonstrated in cat  and primate  that corticothalamic feedback modulates receptive field properties of relay cells in LGN. This evidence has been corroborated by simulation studies [6,7]. We are still far from a thorough understanding of the role and means of cortical modulation of thalamic processing though. We propose a two-pronged approach to further our understanding of visual thalamus. On the one hand, we have derived an abstract mathematical model for signal flow in the early visual pathway, which predicts the effect of feedback on receptive field shape concisely [8,9]. While this model allows for thorough mathematical analysis, it requires rather strong simplifying assumptions, and does not include spike timing at all. We are therefore developing a comprehensive model of the corticothalamic pathway (COTHACO), based on spiking model neurons. We aim to create a model that represents an optimal compromise between the desire for biological realism, and the constraints set by computer resources. This work is supported by the Honda Research Institute Europe.  J. Bullier, Trends Cog Sci 5, 369 (2001).  S.M. Sherman and R.W. Guillery, Exploring the Thalamus (Academic Press, 2001).  K. Funke and F. Wörgötter, Prog Neurobiol 53, 67 (1997).  A.M. Sillito, H.E. Jones, G.L. Gerstein, and D.C. West, Nature, 479 (1994).  X. Xu et al, Vis Neurosci 19, 97 (2002).  K.L. Kirkland and G.L. Gerstein, J Comp Neurosci 7, 255 (1999).  F. Hayot and D. Tranchina, Vis Neurosci 18, 865 (2001).  G.T. Einevoll and H.E. Plesser, Network-Comp Neural 13, 503 (2002).  G.T. Einevoll and H.E. Plesser, in Soc Neurosci Abstr 2003, no
20 Transient perfusion in tumours treated with collagenase or hyaluronidase Ingunn Tufto, David Byberg, Kirsten H. H. Nygaard, Catharina de Lange Davies DEPARTMENT OF PHYSICS, THE NORWEGIAN UNIVERSITY OF SCIENCE AND TECHNOLOGY, TRONDHEIM O-8 A major problem in cancer therapy based on tumour specific macromolecules, is the low uptake of the therapeutic molecule. This is due to low transvascular filtration caused by the high interstitial fluid pressure, and low diffusion through the extracellular matrix. The extracellular matrix consists of a structural protein network of collagen embedded in a hydrofilic gel of gycosaminoglycans. It is not clear whether the collagen network or the glycosaminoglycan gel plays the most important role in limiting the penetration through the extracellular matrix. Collagenase and hyaluronidase, enzymes which degrade the extracellular matrix, have been shown to reduce the interstitial fluid pressure , increase the blood volume and increase the uptake of macromolecules in tumours [2,3]. The purpose of the present work was to investigate whether the increase in blood volume were due to changes in transient perfusion, i.e. vessels that transiently close or open up. Human osteosarcoma xenografts were grown orthotopically around the periosteum of both femurs in athymic mice. Transient perfusion was detected with the double-fluorescent staining method using two fluorescent dyes Hoechst and DiOC 7 (3) . Hoechst was injected intravenously (i.v.) 5 minute prior to collagenase (100 µl, 0.1% solution in PBS) or hyaluronidase (1500 i.e. in PBS), and DiOC 7 (3) was injected 25 minutes thereafter. Frozen tumour sections were analyzed using confocal laser scanning microscopy vessels from each tumour were counted and scored into three different groups depending on whether they were stained with both dyes or only with one of the two dyes. If a vessel was stained with Hoechst only, the vessel had closed before the injection of DiOC 7 (3), and if a vessel was stained with DiOC 7 (3) only, the vessel had opened up after the injection of Hoechst The total fraction of vessels stained with Hoechst only and vessels stained with DiOC 7 (3) only was not statistically different between the untreated tumours and the tumours treated with collagenase or hyaluronidase. There was however a significant increase of vessels stained with DiOC 7 (3) only compared with the decrease of vessels stained with Hoechst only in the tumours treated with collagenase compared with untreated tumours. This was not found with tumours treated with hyaluronidase. A higher fraction of vessels had remained open and a higher fraction of vessels had opened up in collagenase treated tumours compared to untreated ones. This occured more frequently in the central part of the tumours compared to the periphery. The higher amount of vessels that stayed open or opened up in tumours treated with collagenase may explane the increase in blood volume. The differences in transient perfusion between tumours treated with hyaluronidase or collagenase demonstrated that degradation of the structural collagen network surrounding blood vessels has a greater impact on the vascular volume than degradation of the gel of glycosaminoglycans.  C. Brekken et al., Anticancer Res. 20 (5B) (2000)  C. Brekken et al., Anticancer Res. 20 (5B) (2000)  L. Eikenes et al., Cancer Res. Submitted  M.J. Trotter et al., Int. J. Radiat. Oncol. Biol. Phys (1988)
21 Biopolymer assembly and interactions: Nanoscale studies using AFM Signe Danielsen, Marit Sletmoen, Gjertrud Maurstad and Bjørn T. Stokke BIOPHYSICS AND MEDICAL TECHNOLOGY, DEPARTMENT OF PHYSICS, THE NORWEGIAN UNIVERSITY OF SCIENCE AND TECHNOLOGY, TRONDHEIM O-9 The invention of scanning probe microscopes, in particular atomic force microscopes (AFM), yields a technological basis for determination the structure of biological specimens under near physiological conditions and at the same time obtaining a resolution in the range nm. Additionally, elastic properties can be determined, functionalization of AFM probes allow determination of specific interactions, and forced unravelling of higher order structure of single molecules can be used for determination folding imperfections. The structures of polyelectrolyte complexes of semiflexible polyanions with polycations and specific interaction between mannuronan and an epimerase will be used to illustrate some of the potentials of this technique. Manufacturing, control of structure and characterization of nanoparticles are of interest within drug-delivery. Complexes between plasmid DNA and chitosans represent a novel approach to the topic of gene therapy where the purpose is to obtain enhanced cellular uptake, intracellular transport and incorporation in the genome. Chitosans have proven to be promising candidates for obtaining complexes with many beneficial properties, e.g. low cytotoxicity and good biodegradability in combination with documented transfection efficacy. In contrast, viral vectors, which may be very effective, are associated with safety concerns. The structure of nano-sized complexes between a model gene vector and the polycation chitosan were studied using atomic force microscopy. Chitosan/DNA complexes were determined to have a size in the range nm. The importance of the fine structure, and chain length distribution, on the fraction of the characteristic linear and toroidal morphologies was investigated. Polycation induced compaction of DNA was compared with complexation behaviour and other semiflexible, charged biopolymers. Analysis of morphologies of the complexes yielded distribution of asphericity indices allowing classifications into toroidal, linear and globular structures. The data show that the linear fraction is reduced compared to toroids for the stiffer biopolymers, and that the complexation into toroidal structures is not limited to DNA. The elasticity of single alginate chains and their interaction with recombinantly produced enzymes that epimerizes alginate at the polymer level were determined using AFM. The specificity of the interaction was indicated in a competitive assay. The forced dissociation between alginate and epimerase AlgE4 shows a most probable unbinding force in the range 70 to 160 pn depending on the loading rate. This was used as a basis for determination of the distance to the transition state in the unbinding path.
22 Polymer gel signal transducers as interface for optical detection of biological macromolecules in vitro and in vivo Lars Eric Sæther a, Berit Falch b, Dag Roar Hjelme b, Bjørn Torger Stokke a a DEPARTMENT OF PHYSICS, NTNU, TRONDHEIM b OPTOMED, TRONDHEIM O-10 Molecular diagnosis of physiological conditions by concentration determination of a specific set of biological macromolecules directly within a multi-component biological fluid is an appealing approach to avoid extensive isolation and purification steps. This is a challenging task with respect to specificity, sensitivity, dynamic range and kinetics of the signal to be determined. In the clinical chemistry sector there is a definite need of simplifying diagnostic testing and extending test reliability outside the confines of a central laboratory. The ultimate in simplification is integration of biological and measurement elements into a simple monolithic device; a biological sample-interactive phase in close contact with a chemical or physical transducer. Responsive polymer matrices, being intelligent materials having both sensor and transducer functions, are very attractive material candidates for such novel simplified diagnostic test devices. Over the past decades many biospecific responsive polymer gel matrices have been developed for use in conjunction with optical devices to provide novel simplified diagnostic test devices [1-3]. One of the design principles of the biospecific responsive polymer gel is incorporation of both lock and key compounds within the matrix forming a physical crosslink . The external concentration of biomolecules specific for the lock or key will affect the crosslink density with the concomitant change in the equilibrium swelling volume, which can be determined with a precision of 5 nm or better . Molecular design parameters and macroscopic size of the transducer control sensitivity and kinetics. Preliminary results in the diploma work indicate that ph-responsive polymer gels with response time in the order of seconds can be made.  J. Lin, Trends in analytical chemistry (2000)  H.-M. Haake et al., Fresenius J Anal Chem (2000)  R. A. Potyrailo et al., Fresenius J Anal Chem (1998)  M. Shibayama et al., Advances in Polymer Science (1993)  D. R. Hjelme et al., UK and US Patent application, priority date
23 P-1 The impact of the extracellular matrix structure on the uptake of therapeutic macromolecules in solid tumours Hilde Nortvedt Andersen, Arne Erikson, and Catharina de Lange Davies DEPARTMENT OF PHYSICS, THE NORWEGIAN UNIVERSITY OF SCIENCE AND TECHNOLOGY, TRONDHEIM A major problem using tumour specific agents such as monoclonal antibodies recognising tumour-associated antigens and DNA vectors for gene therapy, is the low tumour uptake and heterogeneous distribution of the therapeutic agents. Macromolecules able to cross the capillary wall have to penetrate the extracellular matrix (ECM) by diffusion and convection, driven by concentration-and pressure gradients respectively. Due to the elevated interstitial fluid pressure in tumours, the pressure gradient is low and diffusion becomes the important transport mechanism. The ECM consists of a protein collagen network embedded in a hydrophilic gel of glycosaminoglycans and proteoglycans, and is a major barrier to the delivery of therapeutic macromolecules. We have previously found that the uptake of antibody did not correlate with the amount of ECM constituents such as collagen, hyaluronan, or sulphated glycosaminoglycans (1). In the present work we therefore wanted to study whether the structure of the ECM rather than the amount of its constituents plays the important role in the interstitial transport. To study this, the distribution and diffusion macromolecules in collagen gels were measured. 3-dimentional collagen gels are made by polymerisation of various concentration of collagen (Vitrogen 100) at certain times at 37 C. Combining 3-dimentional reflection and fluorescent laser scanning microscopy, the structure of collagen and the distribution of fluorescent macromolecules were measured. Diffusion of the macromolecules are further measured by fluorescence recovery after photobleaching (FRAP). The reflection microscopy images are analysed to determine the orientation, length and diameter of the collagen fibers. These characteristics of the collagen network are used to correlate the collagen structure with distribution and diffusion of the fluorescent macromolecules. The fluorescent macromolecules applied are dextran-fitc of sizes from 150 kd to kd. Also charged IgG, both cationized and anionized, are used to study the impact of charge of the therapeutic agents on diffusion in collagen gels. 1. Davies, C de L, et al British J Cancer, 85, , 2001
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