Detektion neuroaktiver Substanzen durch Modifikation des Aktivitätsmusters neokortikaler Netzwerke auf Multielektrodenarrays – Charakterisierung und Optimierung eines neuronalen Biosensors

In dieser Arbeit wurden kortikale neuronale Netzwerke auf Multielektrodenarrays auf ihre Tauglichkeit als zellbasiertes Biosensorsystem untersucht. Der Schwerpunkt der pharmakologischen Untersuchungen an den ausgereiften kortikalen Netzwerken lag auf dem Einsatz von Substanzen, welche auf den GABAA-Rezeptor einwirken. Die Modifikation des spontan generierten Aktivitätsmusters ließ dabei Rückschlüsse auf die Wirksamkeit und den Wirkungsmechanismus der Testsubstanzen zu. Ferner war in den meisten Fällen eine Diskriminierung der auf den gleichen Rezeptor einwirkenden Substanzen möglich. Die Analyse der Spikerate und verschiedener auf Bursts beruhender Messparameter machte deutlich, dass die Burstrate bei den extrazellulären Ableitungen auf Netzwerkebene den sensitivsten und verlässlichsten Parameter zum Nachweis der Substanzeffekte darstellte. Durch die Verwendung kortikaler Netzwerke unter optimierten Kulturbedingungen und einer auf das System abgestimmten Analysesoftware konnte die Reproduzierbarkeit und Sensitivität im Vergleich zu anderen Studien deutlich verbessert werden. Um die extrazelluläre Signalableitung von einer möglichst geringen Zellanzahl und damit einem überschaubaren zellulären Netzwerk auf Multielektrodenarrays zu ermöglichen, wurden die Oberflächeneigenschaften der Substrate so modifiziert, dass die Lokalisation der Zellsomata und das Auswachsen der Neurite einer geometrischen Kontrolle unterlag. Die kontrollierte Substratbeschichtung des Adhäsionspromotors Poly-D-Lysin in einem triangulären Muster konnte dabei durch die Methode des Mikrokontaktstempelns realisiert werden. Durch das kontrollierte Zellwachstum konnte die extrazelluläre Ableitung von Netzwerken einer geringen Zelldichte über einen Zeitraum von mehreren Wochen ermöglicht werden. Die Untersuchung struktureller und morphogenetischer Eigenschaften, sowie elektrophysiologische Untersuchungen der strukturierten Netzwerke bewiesen, dass die kontrollierte Substratbeschichtung sich nicht negativ auf das Wachstum, die Synaptogenese und die Funktionalität auswirkte.

Mapping the elastic response of epithelial apical cell membranes suspended across porous array

As the elastic response of cell membranes to mechanical stimuli plays a key role in various cellular processes, novel biophysical strategies to quantify the elasticity of native membranes under physiological conditions at a nanometer scale are gaining interest. In order to investigate the elastic response of apical membranes, elasticity maps of native membrane sheets, isolated from MDCK II (Madine Darby Canine kidney strain II) epithelial cells, were recorded by local indentation with an Atomic Force Microscope (AFM). To exclude the underlying substrate effect on membrane indentation, a highly ordered gold coated porous array with a pore diameter of 1.2 μm was used to support apical membranes. Overlays of fluorescence and AFM images show that intact apical membrane sheets are attached to poly-D-lysine coated porous substrate. Force indentation measurements reveal an extremely soft elastic membrane response if it is indented at the center of the pore in comparison to a hard repulsion on the adjacent rim used to define the exact contact point. A linear dependency of force versus indentation (-dF/dh) up to 100 nm penetration depth enabled us to define an apparent membrane spring constant (kapp) as the slope of a linear fit with a stiffness value of for native apical membrane in PBS. A correlation between fluorescence intensity and kapp is also reported. Time dependent hysteresis observed with native membranes is explained by a viscoelastic solid model of a spring connected to a Kelvin-Voight solid with a time constant of 0.04 s. No hysteresis was reported with chemically fixated membranes. A combined linear and non linear elastic response is suggested to relate the experimental data of force indentation curves to the elastic modulus and the membrane thickness. Membrane bending is the dominant contributor to linear elastic indentation at low loads, whereas stretching is the dominant contributor for non linear elastic response at higher loads. The membrane elastic response was controlled either by stiffening with chemical fixatives or by softening with F-actin disrupters. Overall, the presented setup is ideally suitable to study the interactions of the apical membrane with the underlying cytoskeleton by means of force indentation elasticity maps combined with fluorescence imaging.

Caratterizzazione della superficie di array di nanofili di silicio

Nel primo capitolo viene introdotto lo studio eff�ettuato e descritto un metodo di misure successivo alla caratterizzazione della super�ficie. Nel secondo capitolo vengono descritti i campioni analizzati e, nello speci�fico, la crescita attraverso MaCE dei nanofi�li di silicio. Nel terzo capitolo viene descritto lo strumento AFM utilizzato e la teoria della caratterizzazione alla base dello studio condotto. Nella quarta sezione vengono descritti i risultati ottenuti mentre nelle conclusioni viene tratto il risultato dei valori ottenuti di RMS roughness e roughness exponent.

Hydrodynamic induced by an array of wave energy converters. Experimental and numerical analysis

The thesis analyses the hydrodynamic induced by an array of Wave energy Converters (WECs), under an experimental and numerical point of view. WECs can be considered an innovative solution able to contribute to the green energy supply and –at the same time– to protect the rear coastal area under marine spatial planning considerations. This research activity essentially rises due to this combined concept. The WEC under exam is a floating device belonging to the Wave Activated Bodies (WAB) class. Experimental data were performed at Aalborg University in different scales and layouts, and the performance of the models was analysed under a variety of irregular wave attacks. The numerical simulations performed with the codes MIKE 21 BW and ANSYS-AQWA. Experimental results were also used to calibrate the numerical parameters and/or to directly been compared to numerical results, in order to extend the experimental database. Results of the research activity are summarized in terms of device performance and guidelines for a future wave farm installation. The device length should be “tuned” based on the local climate conditions. The wave transmission behind the devices is pretty high, suggesting that the tested layout should be considered as a module of a wave farm installation. Indications on the minimum inter-distance among the devices are provided. Furthermore, a CALM mooring system leads to lower wave transmission and also larger power production than a spread mooring. The two numerical codes have different potentialities. The hydrodynamics around single and multiple devices is obtained with MIKE 21 BW, while wave loads and motions for a single moored device are derived from ANSYS-AQWA. Combining the experimental and numerical it is suggested –for both coastal protection and energy production– to adopt a staggered layout, which will maximise the devices density and minimize the marine space required for the installation.

Graphene based electrode materials for solar cell and electrochemical oxygen reduction

Diese Arbeit hat viele beispiellose synthetische Ansätze für neuartige Verbundwerkstoffe Graphen-und stickstoffhaltigen graphitischen Materialien erforscht. Die erhaltenen Materialien wurden als den transparenten Elektroden der Solarzellen, die freistehenden Elektroden mit verbesserter mechanischer Festigkeit, und die Kathoden der Brennstoffzellen der Sauerstoffreduktion aufgebracht.rnAlle Ergebnisse haben eindeutig das große Potenzial von Graphen basierenden Materialien und stickstoffhaltigen graphitische Kohlenstoffe als neuartige Elektrodenmaterialien für neue Energie-Geräten demonstriert.

Observability of high density tracing molecular lines in lensed galaxies with the Atacama large millimeter array

Lo scopo di questo lavoro di tesi è indagare le capacità di ALMA di osservare il continuo e le righe molecolari di emissione di HCN, HCO+ e HNC, in galassie lensate ad alto z, nelle prime fasi della loro formazione. Per farlo vengono utilizzate osservazioni ALMA di righe di emissione molecolare, relative a dati pubblici di Ciclo 0. Queste osservazioni vengono utilizzate per simulare in modo realistico l’emissione da parte di galassie ad alto z e la risposta osservativa del telescopio, assumendo diverse possibili geometrie di lensing. Inoltre le recenti osservazioni ALMA sono state utilizzate per aggiornare le relazioni già esistenti tra la luminosità FIR e la luminosità delle righe molecolari. Queste recenti osservazioni con maggiore sensibilità e risoluzione angolare, sembrano essere in buon accordo con le precedenti osservazioni. Per realizzare questo progetto di tesi, sono stati scaricati dall’archivio ALMA i dati non calibrati relativi a due sorgenti ben studiate: NGC1614, rappresentativa delle galassie di tipo starburst ed IRAS 20551-4250, un AGN oscurato. Il processo di calibrazione è stato ripetuto per esaminare le proprietà dei cubi di dati utilizzando il pacchetto Common Astronomy Software Applications (CASA). Le righe spettrali osservate e l’emissione del continuo sono state successivamente estrapolate ad alto z riscalando adeguatamente le distanze, le dimensioni delle sorgenti e le frequenze di emissione. In seguito è stato applicato un modello di lensing gravitazionale basato su quello di Lapi et al. (2012). Sono state considerate diverse configurazioni tra lente e sorgente per ottenere diverse geometrie delle immagini e diversi fattori di amplificazione. Infine stato utilizzato il software CASA per simulare le osservazioni ALMA in modo da verificare le capacità osservative del telescopio al variare della geometria del sistema. Per ogni riga molecolare e per l’emissione del continuo sono state determinate la sensibilit e la risoluzione che possono essere raggiunte con le osservazioni ALMA e sono state analizzate alcune strategie osservative per effettuare survey di righe spettrali in oggetti lensati. Inoltre stata analizzata la possibilit di caratterizzare oggetti starburst ed AGN dai rapporti tra le righe di emissione delle molecole traccianti di alta densit. Le prestazioni di ALMA consentiranno di distinguere e stimare i contributi relativi di SB ed AGN in galassie lensate a z > 2.5, quindi vicine alla loro presunta epoca di formazione (Lapi et al. 2010), in meno di 5 minuti di osservazione per qualsiasi fattore di magnificazione. Nel presente lavoro sono state inoltre discusse alcune strategie osservative per condurre survey o followup per osservare le righe di HCN(4-3), HCO+(4-3) e HNC(4-3) in galassie lensate a redshift 2.5 < z < 3, dimostrando che sono possibili per campioni statisticamente significativi in tempi relativamente brevi.

Non-thermal emission in High Frequency Peaked blazars towards the Square Kilometer Array era

In this thesis work we will explore and discuss the properties of the gamma-ray sources included in the first Fermi-LAT catalog of sources above 10 GeV (1FHL), by considering both blazars and the non negligible fraction of still unassociated gamma-ray sources (UGS, 13%). We perform a statistical analysis of a complete sample of hard gamma-ray sources, included in the 1FHL catalog, mostly composed of HSP blazars, and we present new VLBI observations of the faintest members of the sample. The new VLBI data, complemented by an extensive search of the archives for brighter sources, are essential to gather a sample as large as possible for the assessment of the significance of the correlation between radio and very high energy (E>100 GeV) emission bands. After the characterization of the statistical properties of HSP blazars and UGS, we use a complementary approach, by focusing on an intensive multi-frequency observing VLBI and gamma-ray campaign carried out for one of the most remarkable and closest HSP blazar Markarian 421.

Structure and dynamics of ionic liquid,electrode interfaces

In the early 20th century, Gouy, Chapman, and Stern developed a theory to describe the capacitance and the spatial ion distribution of diluted electrolytes near an electrode. After a century of research, considerable progress has been made in the understanding of the electrolyte/electrode interface. However, its molecular-scale structure and its variation with an applied potential is still under debate. In particular for room-temperature ionic liquids, a new class of solventless electrolytes, the classical theories for the electrical double layer are not applicable. Recently, molecular dynamics simulations and phenomenological theories have attempted to explain the capacitance of the ionic liquid/electrode interface with the molecular-scale structure and dynamics of the ionic liquid near the electrode. rnHowever, experimental evidence is very limited. rnrnIn the presented study, the ion distribution of an ionic liquid near an electrode and its response to applied potentials was examined with sub-molecular resolution. For this purpose, a new sample chamber was constructed, allowing in situ high energy X-ray reflectivity experiments under potential control, as well as impedance spectroscopy measurements. The combination of structural information and electrochmical data provided a comprehensive picture of the electric double layer in ionic liquids. Oscillatory charge density profiles were found, consisting of alternating anion- and cation-enriched layers at both, cathodic and anodic, potentials. This structure was shown to arise from the same ion-ion correlations dominating the liquid bulk structure that were observed as a distinct X-ray diffraction peak. Therefore, existing physically motivated models were refined and verified by comparison with independent measurements. rnrnThe relaxation dynamics of the interfacial structure upon potential variation were studied by time resolved X-ray reflectivity experiments with sub-millisecond resolution. The observed relaxation times during charging/discharging are consistent with the impedance spectroscopy data revealing three processes of vastly different characteristic time-scales. Initially, the ion transport normal to the interface happens on a millisecond-scale. Another 100-millisecond-scale process is associated with molecular reorientation of electrode-adsorbed cations. Further, a minute-scale relaxation was observed, which is tentatively assigned to lateral ordering within the first layer.

Polymer nanoparticles : their functionalization for biomedical applications and dynamics near an electrode

In this work, a method for the functionalization of biocompatible, poly(lactic acid)-based nanoparticles with charged moieties or fluorescent labels is presented. Therefore, a miniemulsion solvent evaporation procedure is used in which prepolymerized poly(L-lactic acid) is used together with a previously synthesized copolymer of methacrylic acid or a polymerizable dye, respectively, and an oligo(lactic acid) macromonomer. Alternatively, the copolymerization has been carried out in one step with the miniemulsion solvent evaporation. The increased stability in salty solutions of the carboxyl-modified nanoparticles compared to nanoparticles consisting of poly(lactic acid) only has been shown in light scattering experiments. The properties of the nanoparticles that were prepared with the separately synthesized copolymer were almost identical to those in which the copolymerization and particle fabrication were carried out simultaneously. During the characterization of the fluorescently labeled nanoparticles, the focus was on the stable bonding between the fluorescent dye and the rest of the polymer chain to ensure that none of it is released from the particles, even after longer storage time or during lengthy experiments. In a fluorescence correlation spectroscopy experiment, it could be shown that even after two weeks, no dye has been released into the solvent. Besides biomedical research for which the above described, functionalized nanoparticles were optimized, nanoparticles also play a role in coating technology. One possibility to fabricate coatings is the electrophoretic deposition of particles. In this process, the mobility of nanoparticles near electrode interfaces plays a crucial role. In this thesis, the nanoparticle mobility has been investigated with resonance enhanced dynamic light scattering (REDLS). A new setup has been developed in which the evanescent electromagnetic eld of a surface plasmon that propagates along the gold-sample interface has been used as incident beam for the dynamic light scattering experiment. The gold layer that is necessary for the excitation of the plasmon doubles as an electrode. Due to the penetration depth of the surface plasmon into the sample layer that is limited to ca. 200 nm, insights on the voltage- and frequency dependent mobility of the nanoparticles near the electrode could be gained. Additionally, simultaneous measurements at four different scattering angles can be carried out with this setup, therefore the investigation of samples undergoing changes is feasible. The results were discussed in context with the mechanisms of electrophoretic deposition.

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

Thin-Film Sparse Boundary Array Design for Passive Acoustic Mapping During Ultrasound Therapy

A new 2-D hydrophone array for ultrasound therapy monitoring is presented, along with a novel algorithm for passive acoustic mapping using a sparse weighted aperture. The array is constructed using existing polyvinylidene fluoride (PVDF) ultrasound sensor technology, and is utilized for its broadband characteristics and its high receive sensitivity. For most 2-D arrays, high-resolution imagery is desired, which requires a large aperture at the cost of a large number of elements. The proposed array's geometry is sparse, with elements only on the boundary of the rectangular aperture. The missing information from the interior is filled in using linear imaging techniques. After receiving acoustic emissions during ultrasound therapy, this algorithm applies an apodization to the sparse aperture to limit side lobes and then reconstructs acoustic activity with high spatiotemporal resolution. Experiments show verification of the theoretical point spread function, and cavitation maps in agar phantoms correspond closely to predicted areas, showing the validity of the array and methodology.

A 20-channel receive-only mouse array coil for a 3 T clinical MRI system

A 20-channel phased-array coil for MRI of mice has been designed, constructed, and validated with bench measurements and high-resolution accelerated imaging. The technical challenges of designing a small, high density array have been overcome using individual small-diameter coil elements arranged on a cylinder in a hexagonal overlapping design with adjacent low impedance preamplifiers to further decouple the array elements. Signal-to-noise ratio (SNR) and noise amplification in accelerated imaging were simulated and quantitatively evaluated in phantoms and in vivo mouse images. Comparison between the 20-channel mouse array and a length-matched quadrature driven small animal birdcage coil showed an SNR increase at the periphery and in the center of the phantom of 3- and 1.3-fold, respectively. Comparison with a shorter but SNR-optimized birdcage coil (aspect ratio 1:1 and only half mouse coverage) showed an SNR gain of twofold at the edge of the phantom and similar SNR in the center. G-factor measurements indicate that the coil is well suited to acquire highly accelerated images.