Microarray based real-time analysis of nucleic acid hybridization kinetics and thermodynamics

Ziel dieser Dissertation ist die experimentelle Charakterisierung und quantitative Beschreibung der Hybridisierung von komplementären Nukleinsäuresträngen mit oberflächengebundenen Fängermolekülen für die Entwicklung von integrierten Biosensoren. Im Gegensatz zu lösungsbasierten Verfahren ist mit Microarray Substraten die Untersuchung vieler Nukleinsäurekombinationen parallel möglich. Als biologisch relevantes Evaluierungssystem wurde das in Eukaryoten universell exprimierte Actin Gen aus unterschiedlichen Pflanzenspezies verwendet. Dieses Testsystem ermöglicht es, nahe verwandte Pflanzenarten auf Grund von geringen Unterschieden in der Gen-Sequenz (SNPs) zu charakterisieren. Aufbauend auf dieses gut studierte Modell eines House-Keeping Genes wurde ein umfassendes Microarray System, bestehend aus kurzen und langen Oligonukleotiden (mit eingebauten LNA-Molekülen), cDNAs sowie DNA und RNA Targets realisiert. Damit konnte ein für online Messung optimiertes Testsystem mit hohen Signalstärken entwickelt werden. Basierend auf den Ergebnissen wurde der gesamte Signalpfad von Nukleinsärekonzentration bis zum digitalen Wert modelliert. Die aus der Entwicklung und den Experimenten gewonnen Erkenntnisse über die Kinetik und Thermodynamik von Hybridisierung sind in drei Publikationen zusammengefasst die das Rückgrat dieser Dissertation bilden. Die erste Publikation beschreibt die Verbesserung der Reproduzierbarkeit und Spezifizität von Microarray Ergebnissen durch online Messung von Kinetik und Thermodynamik gegenüber endpunktbasierten Messungen mit Standard Microarrays. Für die Auswertung der riesigen Datenmengen wurden zwei Algorithmen entwickelt, eine reaktionskinetische Modellierung der Isothermen und ein auf der Fermi-Dirac Statistik beruhende Beschreibung des Schmelzüberganges. Diese Algorithmen werden in der zweiten Publikation beschrieben. Durch die Realisierung von gleichen Sequenzen in den chemisch unterschiedlichen Nukleinsäuren (DNA, RNA und LNA) ist es möglich, definierte Unterschiede in der Konformation des Riboserings und der C5-Methylgruppe der Pyrimidine zu untersuchen. Die kompetitive Wechselwirkung dieser unterschiedlichen Nukleinsäuren gleicher Sequenz und die Auswirkungen auf Kinetik und Thermodynamik ist das Thema der dritten Publikation. Neben der molekularbiologischen und technologischen Entwicklung im Bereich der Sensorik von Hybridisierungsreaktionen oberflächengebundener Nukleinsäuremolekülen, der automatisierten Auswertung und Modellierung der anfallenden Datenmengen und der damit verbundenen besseren quantitativen Beschreibung von Kinetik und Thermodynamik dieser Reaktionen tragen die Ergebnisse zum besseren Verständnis der physikalisch-chemischen Struktur des elementarsten biologischen Moleküls und seiner nach wie vor nicht vollständig verstandenen Spezifizität bei.

Design of a semi-implantable hearing device for direct acoustic cochlear stimulation

A new hearing therapy based on direct acoustic cochlear stimulation was developed for the treatment of severe to profound mixed hearing loss. The device efficacy was validated in an initial clinical trial with four patients. This semi-implantable investigational device consists of an externally worn audio processor, a percutaneous connector, and an implantable microactuator. The actuator is placed in the mastoid bone, right behind the external auditory canal. It generates vibrations that are directly coupled to the inner ear fluids and that, therefore, bypass the external and the middle ear. The system is able to provide an equivalent sound pressure level of 125 dB over the frequency range between 125 and 8000 Hz. The hermetically sealed actuator is designed to provide maximal output power by keeping its dimensions small enough to enable implantation. A network model is used to simulate the dynamic characteristics of the actuator to adjust its transfer function to the characteristics of the middle ear. The geometry of the different actuator components is optimized using finite-element modeling.

Uncovering the dynamics of cardiac systems using stochastic pacing and frequency domain analyses

Alternans of cardiac action potential duration (APD) is a well-known arrhythmogenic mechanism which results from dynamical instabilities. The propensity to alternans is classically investigated by examining APD restitution and by deriving APD restitution slopes as predictive markers. However, experiments have shown that such markers are not always accurate for the prediction of alternans. Using a mathematical ventricular cell model known to exhibit unstable dynamics of both membrane potential and Ca2+ cycling, we demonstrate that an accurate marker can be obtained by pacing at cycle lengths (CLs) varying randomly around a basic CL (BCL) and by evaluating the transfer function between the time series of CLs and APDs using an autoregressive-moving-average (ARMA) model. The first pole of this transfer function corresponds to the eigenvalue (λalt) of the dominant eigenmode of the cardiac system, which predicts that alternans occurs when λalt≤−1. For different BCLs, control values of λalt were obtained using eigenmode analysis and compared to the first pole of the transfer function estimated using ARMA model fitting in simulations of random pacing protocols. In all versions of the cell model, this pole provided an accurate estimation of λalt. Furthermore, during slow ramp decreases of BCL or simulated drug application, this approach predicted the onset of alternans by extrapolating the time course of the estimated λalt. In conclusion, stochastic pacing and ARMA model identification represents a novel approach to predict alternans without making any assumptions about its ionic mechanisms. It should therefore be applicable experimentally for any type of myocardial cell.

IMPACT OF ENGINE CALIBRATION ON PM OXIDATION IN A CATALYZED PARTICULATE FILTER OVER A TRANSIENT CYCLE: A MODELLING STUDY

The combustion strategy in a diesel engine has an impact on the emissions, fuel consumption and the exhaust temperatures. The PM mass retained in the CPF is a function of NO2 and PM concentrations in addition to the exhaust temperatures and the flow rates. Thus the engine combustion strategy affects exhaust characteristics which has an impact on the CPF operation and PM mass retained and oxidized. In this report, a process has been developed to simulate the relationship between engine calibration, performance and HC and PM oxidation in the DOC and CPF respectively. Fuel Rail Pressure (FRP) and Start of Injection (SOI) sweeps were carried out at five steady state engine operating conditions. This data, along with data from a previously carried out surrogate HD-FTP cycle [1], was used to create a transfer function model which estimates the engine out emissions, flow rates, temperatures for varied FRP and SOI over a transient cycle. Four different calibrations (test cases) were considered in this study, which were simulated through the transfer function model and the DOC model [1, 2]. The DOC outputs were then input into a model which simulates the NO2 assisted and thermal PM oxidation inside a CPF. Finally, results were analyzed as to how engine calibration impacts the engine fuel consumption, HC oxidation in the DOC and the PM oxidation in the CPF. Also, active regeneration for various test cases was simulated and a comparative analysis of the fuel penalties involved was carried out.

Precise Near-to-Head Acoustics with Binaural Synthesis

For enhanced immersion into a virtual scene more than just the visual sense should be addressed by a Virtual Reality system. Additional auditory stimulation appears to have much potential, as it realizes a multisensory system. This is especially useful when the user does not have to wear any additional hardware, e.g., headphones. Creating a virtual sound scene with spatially distributed sources requires a technique for adding spatial cues to audio signals and an appropriate reproduction. In this paper we present a real-time audio rendering system that combines dynamic crosstalk cancellation and multi-track binaural synthesis for virtual acoustical imaging. This provides the possibility of simulating spatially distributed sources and, in addition to that, near-to-head sources for a freely moving listener in room-mounted virtual environments without using any headphones. A special focus will be put on near-to-head acoustics, and requirements in respect of the head-related transfer function databases are discussed.

A chrysophyte stomatocyst-based reconstruction of cold-season air temperature from Alpine Lake Silvaplana (AD 1500–2003); methods and concepts for quantitative inferences

Relatively little is known about past cold-season temperature variability in high-Alpine regions because of a lack of natural cold-season temperature proxies as well as under-representation of high-altitude sites in meteorological, early-instrumental and documentary data sources. Recent studies have shown that chrysophyte stomatocysts, or simply cysts (sub-fossil algal remains of Chrysophyceae and Synurophyceae), are among the very few natural proxies that can be used to reconstruct cold-season temperatures. This study presents a quantitative, high-resolution (5-year), cold-season (Oct–May) temperature reconstruction based on sub-fossil chrysophyte stomatocysts in the annually laminated (varved) sediments of high-Alpine Lake Silvaplana, SE Switzerland (1,789 m a.s.l.), since AD 1500. We first explore the method used to translate an ecologically meaningful variable based on a biological proxy into a simple climate variable. A transfer function was applied to reconstruct the ‘date of spring mixing’ from cyst assemblages. Next, statistical regression models were tested to convert the reconstructed ‘dates of spring mixing’ into cold-season surface air temperatures with associated errors. The strengths and weaknesses of this approach are thoroughly tested. One much-debated, basic assumption for reconstructions (‘stationarity’), which states that only the environmental variable of interest has influenced cyst assemblages and the influence of confounding variables is negligible over time, is addressed in detail. Our inferences show that past cold-season air-temperature fluctuations were substantial and larger than those of other temperature reconstructions for Europe and the Alpine region. Interestingly, in this study, recent cold-season temperatures only just exceed those of previous, multi-decadal warm phases since AD 1500. These findings highlight the importance of local studies to assess natural climate variability at high altitudes.

A chrysophyte-based quantitative reconstruction of winter severity from varved lake sediments in NE Poland during the past millennium and its relationship to natural climate variability

Chrysophyte cysts are recognized as powerful proxies of cold-season temperatures. In this paper we use the relationship between chrysophyte assemblages and the number of days below 4 °C (DB4 °C) in the epilimnion of a lake in northern Poland to develop a transfer function and to reconstruct winter severity in Poland for the last millennium. DB4 °C is a climate variable related to the length of the winter. Multivariate ordination techniques were used to study the distribution of chrysophytes from sediment traps of 37 low-land lakes distributed along a variety of environmental and climatic gradients in northern Poland. Of all the environmental variables measured, stepwise variable selection and individual Redundancy analyses (RDA) identified DB4 °C as the most important variable for chrysophytes, explaining a portion of variance independent of variables related to water chemistry (conductivity, chlorides, K, sulfates), which were also important. A quantitative transfer function was created to estimate DB4 °C from sedimentary assemblages using partial least square regression (PLS). The two-component model (PLS-2) had a coefficient of determination of View the MathML sourceRcross2 = 0.58, with root mean squared error of prediction (RMSEP, based on leave-one-out) of 3.41 days. The resulting transfer function was applied to an annually-varved sediment core from Lake Żabińskie, providing a new sub-decadal quantitative reconstruction of DB4 °C with high chronological accuracy for the period AD 1000–2010. During Medieval Times (AD 1180–1440) winters were generally shorter (warmer) except for a decade with very long and severe winters around AD 1260–1270 (following the AD 1258 volcanic eruption). The 16th and 17th centuries and the beginning of the 19th century experienced very long severe winters. Comparison with other European cold-season reconstructions and atmospheric indices for this region indicates that large parts of the winter variability (reconstructed DB4 °C) is due to the interplay between the oscillations of the zonal flow controlled by the North Atlantic Oscillation (NAO) and the influence of continental anticyclonic systems (Siberian High, East Atlantic/Western Russia pattern). Differences with other European records are attributed to geographic climatological differences between Poland and Western Europe (Low Countries, Alps). Striking correspondence between the combined volcanic and solar forcing and the DB4 °C reconstruction prior to the 20th century suggests that winter climate in Poland responds mostly to natural forced variability (volcanic and solar) and the influence of unforced variability is low.

Foraminifer counts in core JM01-1199, and review of radiocarbon ages in the Norwegian-Barents Sea-Svalbard margin

We compare six high-resolution Holocene, sediment cores along a S-N transect on the Norwegian-Svalbard continental margin from ca 60°N to 77.4°N, northern North Atlantic. Planktonic foraminifera in the cores were investigated to show the changes in upper surface and subsurface water mass distribution and properties, including summer sea-surface temperatures (SST). The cores are located below the axis of the Norwegian Current and the West Spitsbergen Current, which today transport warm Atlantic Water to the Arctic. Sediment accumulation rates are generally high at all the core sites, allowing for a temporal resolution of 10-102 years. SST is reconstructed using different types of transfer functions, resulting in very similar SST trends, with deviations of no more than +- 1.0/1.5 °C. A transfer function based on the maximum likelihood statistical approach is found to be most relevant. The reconstruction documents an abrupt change in planktonic foraminiferal faunal composition and an associated warming at the Younger Dryas-Preboreal transition. The earliest part of the Holocene was characterized by large temperature variability, including the Preboreal Oscillations and the 8.2 k event. In general, the early Holocene was characterized by SSTs similar to those of today in the south and warmer than today in the north, and a smaller S-N temperature gradient (0.23 °C/°N) compared to the present temperature gradient (0.46 °C/°N). The southern proxy records (60-69°N) were more strongly influenced by slightly cooler subsurface water probably due to the seasonality of the orbital forcing and increased stratification due to freshening. The northern records (72-77.4°N) display a millennial-scale change associated with reduced insolation and a gradual weakening of the North Atlantic thermohaline circulation (THC). The observed northwards amplification of the early Holocene warming is comparable to the pattern of recent global warming and future climate modelling, which predicts greater warming at higher latitudes. The overall trend during mid and late Holocene was a cooling in the north, stable or weak warming in the south, and a maximum S-N SST gradient of ca 0.7 °C/°N at 5000 cal. years BP. Superimposed on this trend were several abrupt temperature shifts. Four of these shifts, dated to 9000-8000, 5500-3000 and 1000 and ~400 cal. years BP, appear to be global, as they correlate with periods of global climate change. In general, there is a good correlation between the northern North Atlantic temperature records and climate records from Norway and Svalbard.

Age determination and sea-surface reconstruction of sediment cores from the Norwegian Sea

Polycystine radiolarians are used to reconstruct summer sea surface temperatures (SSSTs) for the Late Pleistocene-Holocene (600-13,400 14C years BP) in the Norwegian Sea. At 13,200 14C years BP, the SSST was close to the average Holocene SSST (~12°C). It then gradually dropped to 7.1°C in the Younger Dryas. Near the Younger Dryas-Holocene transition (~10,000 14C years BP), the SSST increased 5°C in about 530 years. Four abrupt cooling events, with temperature drops of up to 2.1°C, are recognized during the Holocene: at 9340, 7100 ("8200 calendar years event"), 6400 and 1650 14C years BP. Radiolarian SSSTs and the isotopic signal from the GISP2 ice core are strongly coupled, stressing the importance of the Norwegian Sea as a mediator of heat/precipitation exchange between the North Atlantic, the atmosphere, and the Greenland ice sheet. Radiolarian and diatom-derived SSSTs display similarities, with the former not showing the recently reported Holocene cooling trend.

Stable isotope record of planktonic foraminifera from sediment core MD96-2085

A high-resolution (~4-5cm/kyr) giant piston core record (MD962085) retrieved during an IMAGES II-NAUSICAA cruise from the continental slope of the southeast Atlantic Ocean reveals striking variations in planktonic foraminifer faunal abundances and sea-surface temperatures (SST) during the past 600 000 yr. The location and high-quality sedimentary record of the core provide a good opportunity to assess the variability of the Benguela Current system and associated important features of the ocean-climate system in the southeast Atlantic. The planktonic foraminifer faunal abundances of the core are dominated by three assemblages: (1) Neogloboquadrina pachyderma (right coiling) + Neogloboquadrina dutertrei, (2) Globigerina bulloides, and (3) Globorotalia inflata. The assemblage of N. pachyderma (right coiling) + N. dutertrei shows distinctive abundance changes which are nearly in-phase with glacial-interglacial variations. The high abundances of this assemblage are associated with major glacial conditions, possibly representing low SST/high nutrient level conditions in the southwestern Africa margin. In contrast, the G. bulloides and G. inflata assemblages show greater high-frequency abundance change patterns, which are not parallel to the glacial-interglacial changes. These patterns may indicate rapid oceanic frontal movements from the south, and a rapid change in the intensity of the Benguela upwelling system from the east. A single episode of maximum abundances of a polar water species N. pachyderma (left coiling) occurred in the beginning of stage 9 (~340-330 kyr). The event of the maximum occurrence of this species shown in this record may indicate instability in the Benguela coastal upwelling, or the Antarctic polar front zone position. A winter season SST estimate using transfer function techniques for this record shows primarily glacial-interglacial variations. The SST is maximal during the transitions from the major glacial to interglacial stages (Terminations I, II, IV, V), and is associated with the abundance maxima of a warm water species indicator Globigerinoides ruber. Cross-spectral analyses of the SST record and the SPECMAP stack reveal statistically significant concentrations of variance and coherencies in three major orbital frequency bands. The SST precedes changes in the global ice volume in all orbital frequency bands, indicating a dominant southern Hemispheric climate effect over the Benguela Current region in the southeast Atlantic.

Paleoproductivity reconstruction for the eastern equatorial Pacific

Productivity at six core locations in the eastern equatorial Pacific (EEP) was reconstructed with a benthic foraminiferal transfer function. The core records show strong regionality, especially where affected by Peru margin upwelling of deeper Equatorial Undercurrent Water (EUC) (originally coming from the subantarctic). This "Peru margin" record differs from that seen along the equator where divergence leads to shallow upwelling, and it is generally inverse to that seen in cores outside the areas of equatorial upwelling. Principal components analysis shows that the main productivity pattern correlates well to the global oxygen isotope record and has lowest values during isotope stages 2 and 4. In addition to this, equatorial cores show a higher frequency pattern of variation which becomes much more pronounced during MIS 3 and 2. The reconstructions based on benthic foraminifera were tested against those from nonaccumulation rate based inorganic chemical proxies of export production. These were found to correlate well in the region influenced by Peru upwelling, and also to share common features for sites along the equator. All the nonaccumulation rate based paleotracers are consistent with one another and differ from accumulation rate derived proxies. The differences between the two classes of paleotracers may result from uncertainties in calculating actual biogenic fluxes since 230Th-normalized results conform more to those we obtained. Analysis of planktonic carbon isotope values for the EEP, and their comparison to the record of the Pacific subantarctic, indicates that the subantarctic contribution to the EUC was reduced during MIS 3 and 2.

Age determination of sediment core SO75-26KL, Portuguese margin

A benthic isotope record has been measured for core SO75-26KL from the upper Portuguese margin (1099 m water depth) to monitor the response of thermohaline overturn in the North Atlantic during Heinrich events. Evaluating benthic delta18O in TS diagrams in conjunction with equilibrium deltac fractionation implies that advection of Mediterranean outflow water (MOW) to the upper Portuguese margin was significantly reduced during the last glacial (< 15% compared to 30% today). The benthic isotope record along core SO75-26KL therefore primarily monitors variability of glacial North Atlantic conveyor circulation. The 14C-accelerator mass spectrometry ages of 13.54±.07 and 20.46±.12 ka for two ice-rafted detritus (IRD) layers in the upper core section and an interpolated age of 36.1 ka for a third IRD layer deeper in the core are in the range of published 14C ages for Heinrich events H1, H2, and H4. Marked depletion of benthic delta13C by 0.7-1.1 per mil during the Heinrich events suggests reduced thermohaline overturn in the North Atlantic during these events. Close similarity between meltwater patterns (inferred from planktonic delta18O) at Site 609 and ventilation patterns (inferred from benthic delta13C) in core SO75-26KL implies coupling between thermohaline overturn and surface forcing, as is also suggested by ocean circulation models. Benthic delta13C starts to decrease 1.5-2.5 kyr before Heinrich events Hl and H4, fully increased values are reached 1.5-3 kyr after the events, indicating a successive slowdown of thermohaline circulation well before the events and resumption of the conveyor's full strength well after the events. Benthic delta13C changes in the course of the Heinrich events show subtle maxima and minima suggesting oscillatory behavior of thermohaline circulation, a distinct feature of thermohaline instability in numerical models. Inferrred gradual spin-up of thermohaline circulation after Hl and H4 is in contrast to abrupt wanning in the North Atlantic region that is indicated by sudden increases in Greenland ice core delta18O and in marine faunal records from the northern North Atlantic. From this we infer that thermohaline circulation can explain only in part the rapid climatic oscillations seen in glacial sections of the Greenland ice core record.

Benthic and planktonic foraminifera of sediment core GIK17957-2

A detailed age model for core 17957-2 of the southern South China Sea was developed based on delta18O, coarse fraction, magnetostratigraphy, and biostratigraphy for the last 1500 kyr. The delta18O record has clear ~100-kyr cycles after the Mid-Pleistocene Revolution (MPR) at the entrance of marine isotopic stage (MIS) 22. Planktonic foraminifera responded to the MPR immediately, showing the increased sea surface temperature (SST) and dissolution after the MPR. Benthic foraminifera did not respond to it until the Brunhes/Matuyama boundary. Since the MPR, the depth of thermocline gradually became shallower until MISs 6-5. This major change within MISs 6-5 was also reflected in the decreased SSTs and increased productivity and Deep Water Mass. Thus two major Pleistocene paleoceanographic changes were found: One was around the MPR; the other occurred within MISs 6-5, which speculatively might be ascribed to the reorganization of surface and deep circulation, possibly induced by tectonic forces.

Diatom abundance and paleo-temperature reconstruction for sediment cores from the southeast Norwegian Sea

Although the pulsating nature and the abruptness of the last deglaciation are well documented in marine and land records, very few marine records have so far been able to capture the high-frequency climatic changes recorded in the Greenland ice core Dye 3. We studied high-resolution sediment cores from SE Norwegian Sea, which display a detailed climatic record during the last deglaciation comparable to that of Dye 3. Accelerator mass spectrometry age control of the cores enables us to correlate this record in detail with continental records. The results indicate that the surface waters of the SE Norwegian Sea were seasonally ice free after 13,400 B.P. The Bølling/Allerød interstadial complex (13,200-11,200 B.P.) was a climatically unstable period with changing Arctic-Subarctic conditions. This period was punctuated by four progressively more severe sea surface temperature (SST) minima: between 12,900-12,800 B.P. (BCP I); 12,500-12,400 B.P. (BCP II); 12,300-12,000 B.P. (OD I); and 11,800-11,500 B.P. (OD II). The Younger Dryas (YD) (11,200-10,200 B.P.) represents the severest and most prolonged cold episode of this series of climatic deteriorations. It was bounded by very rapid SST changes and characterized by Arctic-Polar conditions. The first true warm Atlantic water incursion to the SE Norwegian Sea took place around 10,100 B.P., followed by a brief cooler condition between 9900-9600 B.P. (YD II). The early Holocene climatic optimum occurred between 8000-5000 B.P. A conceptual model is proposed where meltwater fluxes are suggested to cause the observed instability in the SST record.