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The PMCA assay was optimized for adaptation to low level detection of PrPSc in hamster plasma. Evaluation of numerous key variables of the PMCA assay led to an optimized protocol capable of ~3 log10 amplification after 32 cycles (two 16 hour rounds). When commercially purchased normal hamster plasma was added to the PMCA reaction an accentuation in PrPSc amplification was observed (>6.75 log10 after 32 cycles). Only con-specific plasma appeared to enhance the conversion of PrPC to PrPSc, suggesting that a species-specific co-factor may be involved in assembly of protein aggregates. Serial PMCA in the presence of low level (10%) contiguous conspecific plasma resulted in the generation of de novo PrPSc after several rounds of PMCA. Although plasma significantly accentuated PrPSc amplification by PMCA, the formation of de novo PrPSc interfered with the ability of using the PMCA assay to detect prion infections in hamsters experimentally infected with 263K scrapie. / Physiology, Cell and Developmental Biology

Recent proceedings in biotechnology have enabled completely new avenues in life science research to be explored. By allowing increased parallelization an ever-increasing complexity of cell samples or experiments can be investigated in shorter time and at a lower cost. This facilitates for example large-scale efforts to study cell heterogeneity at the single cell level, by analyzing cells in parallel that also can include global genomic analyses. The work presented in this thesis focuses on massively parallel analysis of cells or nucleic acid samples, demonstrating technology developments in the field as well as use of the technology in life sciences. In stem cell research issues such as cell morphology, cell differentiation and effects of reprogramming factors are frequently studied, and to obtain information on cell heterogeneity these experiments are preferably carried out on single cells. In paper I we used a high-density microwell device in silicon and glass for culturing and screening of stem cells. Maintained pluripotency in stem cells from human and mouse was demonstrated in a screening assay by antibody staining and the chip was furthermore used for studying neural differentiation. The chip format allows for low sample volumes and rapid high-throughput analysis of single cells, and is compatible with Fluorescence Activated Cell Sorting (FACS) for precise cell selection. Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences by constantly producing increasing amounts of data from one sequencing run. However, the reagent costs and labor requirements in current massively parallel sequencing protocols are still substantial. In paper II-IV we have focused on flow-sorting techniques for improved sample preparation in bead-based massive sequencing platforms, with the aim of increasing the amount of quality data output, as demonstrated on the Roche/454 platform. In paper II we demonstrate a rapid alternative to the existing shotgun sample titration protocol and also use flow-sorting to enrich for beads that carry amplified template DNA after emulsion PCR, thus obtaining pure samples and with no downstream sacrifice of DNA sequencing quality. This should be seen in comparison to the standard 454-enrichment protocol, which gives rise to varying degrees of sample purity, thus affecting the sequence data output of the sequencing run. Massively parallel sequencing is also useful for deep sequencing of specific PCR-amplified targets in parallel. However, unspecific product formation is a common problem in amplicon sequencing and since these shorter products may be difficult to fully remove by standard procedures such as gel purification, and their presence inevitably reduces the number of target sequence reads that can be obtained in each sequencing run. In paper III a gene-specific fluorescent probe was used for target-specific FACS enrichment to specifically enrich for beads with an amplified target gene on the surface. Through this procedure a nearly three-fold increase in fraction of informative sequences was obtained and with no sequence bias introduced. Barcode labeling of different DNA libraries prior to pooling and emulsion PCR is standard procedure to maximize the number of experiments that can be run in one sequencing lane, while also decreasing the impact of technical noise. However, variation between libraries in quality and GC content affects amplification efficiency, which may result in biased fractions of the different libraries in the sequencing data. In paper IV barcode specific labeling and flow-sorting for normalization of beads with different barcodes on the surface was used in order to weigh the proportion of data obtained from different samples, while also removing mixed beads, and beads with no or poorly amplified product on the surface, hence also resulting in an increased sequence quality. In paper V, cell heterogeneity within a human being is being investigated by low-coverage whole genome sequencing of single cell material. By focusing on the most variable portion of the human genome, polyguanine nucleotide repeat regions, variability between different cells is investigated and highly variable polyguanine repeat loci are identified. By selectively amplifying and sequencing polyguanine nucleotide repeats from single cells for which the phylogenetic relationship is known, we demonstrate that massively parallel sequencing can be used to study cell-cell variation in length of these repeats, based on which a phylogenetic tree can be drawn. / QC 20111031

L'objectif de cette thèse est d'étudier une technique de combinaison cohérente de faisceaux : l'amplification paramétrique optique (OPA) à multiple pompes. Cette technique permet de transférer instantanément l'énergie de nombreuses pompes en un unique faisceau signal sans stockage d'énergie, et ainsi s'affranchissant d'effets thermiques dans le milieu amplificateur. Ceci peut s'avérer intéressant pour combiner l'énergie de multiples lasers à fibre et réaliser l'amplification à forte cadence de lasers très énergétiques ou d'impulsions à spectre large. A l'aide d'un code de calcul général et d'une étude expérimentale utilisant comme cristal non linéaire du BBO ou du LBO, nous calculons dans un premier temps la localisation des pompes autour du signal à amplifier, ainsi que les tolérances angulaires correspondantes qui déterminent la criticité d'alignement d'une telle configuration. Nous nous intéressons ensuite aux mécanismes de recombinaisons parasites entre une pompe et l'idler correspondant à une autre pompe. Après avoir démontré expérimentalement que ces recombinaisons peuvent dégrader les caractéristiques spatiales et spectrales du signal amplifié, nous montrons qu'il est possible d'éliminer ces risques de couplages néfastes en écartant suffisamment les pompes entre elles. Une modélisation originale de l'OPA multi-pompes suggère de relier ces phénomènes parasites aux effets des réseaux résultant des interactions entre les différentes pompes. La dernière partie présente l'expérience d'OPA à 5 pompes qui nous a permis d'atteindre un rendement de transfert énergétique des pompes vers le signal de 27%, et obtenir ainsi un signal plus énergétique que chaque pompe prise séparément. / This work deals with a technique of combination of coherent beams: Optical Parametric Amplification (OPA) with Multiple Pumps. This technique is used to instantly transfer the energy of several pumps on one beam, without energy storage and thus avoiding thermal effects in the amplifying media. It can be useful to combine energy of numerous fiber lasers and to amplifiy with a high repetition rate very high energy lasers or broadband pulses. With a numerical and experimental study using BBO and LBO as nonlinear crystal, we determine how to dispose the pumps around the signal and the corresponding angular tolerances of such set up. Then we focus our attention on recombining mechanisms between a pump and a non-corresponding idler. We demonstrate experimentally that these cascading effects may decrease the spatial and spectral quality of the amplified signal, and that these phenomena can be avoided with a minimum angle between the different pumps. A novel modelling of multi-pumps OPA links these cascading effects to the gratings generated by the interaction between the pumps. The last part presents a 5 pump OPA experiment. We achieve a pump-to-signal efficiency of 27% and so that a signal more powerful than each pump is obtained.

Les impulsions femtosecondes (fs) sont employées pour réaliser des interactions lumière matière athermiques intéressant aussi bien les mondes industriel, médical que scientifique.Des lasers avec toujours plus de puissance crête (P c ) à des cadences toujours plus élevées sont requis. Les sources à fibre dopée ytterbium ont pour cela un potentiel important. Or, la durée des impulsions amplifiées demeure élevée (~ 300 fs) en raison du rétrécissement du spectre amplifié pour de forts niveaux de gain, limitant la valeur de P c accessible. L’amplification avec division spectrale à travers un réseau d’amplificateurs fibrés et la synthèse d’impulsions fs par recombinaison spectrale cohérente est proposée comme solution. Les composantes spectrales sont amplifiées séparément en parallèle avant d’être réassemblées en un seul faisceau. La gestion des relations de phase entre les rayonnements issus des voies assurent la reconstruction de l’impulsion après amplification. Différentes architectures sont considérées.Après avoir choisi et dimensionné l’une d’entre elles, nous avons réalisé l’amplification et la synthèse d’impulsions de 280 fs à travers 12 guides non couplés d’une fibre multicœur, sans étireur/compresseur. Nous avons mis en évidence le gain en puissance de cette architecture par rapport à un amplificateur monovoie, proportionnel au carré du nombre de voies mises enjeu. La compatibilité de ce montage avec l’amplification d’impulsions large bande (≈ 40 nm)a été prouvée. En perspective, les performances énergétiques accessibles et la transposition du schéma d’amplification aux oscillateurs en vue de produire des impulsions fs large bande à haute énergie sont discutées. / Femtosecond pulses (fs) are used to produce no thermal light matter interactions which areinteresting for industrial, medical, or scientific activities. Lasers producing higher peak powerat a higher repetition rate are required. Ytterbium doped fiber sources are good candidates.However, pulse duration is still high (~ 300 fs) owing to spectral narrowing at high gainlevels. Peak power is also limited. Amplification in an array of amplifiers with spectralsplitting and fs pulse synthesis by coherent spectral combining is proposed as a solution.Spectral components are separately amplified before to coherently recombine the amplifieroutputs in a single beam. Phase management of the radiations from different amplifiers leadsto short pulse synthesis. Different setups are considered. After the choice and the gauging ofone of them, we amplified and synthesized 280 fs pulses through 12 uncoupled cores of amulticore fiber, without stretcher/compressor devices. We demonstrated the powerenhancement of this setup compared to a single amplifier, proportional to the square of thenumber of amplifier used. Compatibility of the setup with broadband amplification (≈ 40 nm)was demonstrated. In prospects, performance scaling in terms of peak power are in a first timedevelopped. The conception of an oscillator based of this amplification scheme to produce fsbroadband and energetic pulses is proposed in a second time.

L'objet de cette thèse vise à montrer expérimentalement l'amplification dans le domaine terahertz d'échantillons à base de puits quantiques en nitrure de gallium (GaN), maintenus à basses températures (< 100 K), grâce au mécanisme physique de "résonance du temps de transit des phonons optiques". Po ur ce faire, un banc expérimental permettant de mesurer le spectre en transmission des échantillons dans différentes gammes de fréquences (0.220-0.325, 0.843-1.100 et 0.7-1.7 THz suivant la source utilisée), et sous différentes conditions (température, orientation, champ électrique appliqué) a été développé. Un deuxième banc, servant à mesurer les caractéristiques courant-tension en régime continu ou pulsé a également été réalisé en vue de l'étude électrique des composants à basses températures. Les résultats montrent l'existence d'un effet conséquent sur le spectre de transmission au fur et à mesure que le champ électrique appliqué augmente. / The objective of this thesis is to experimentally demonstrate the amplification in the terahertz frequency domain by quantum wells of gallium nitride (GaN), maintained at low temperatures (< 100 K), thanks to the so called "optical phonon transit time resonance" mechanism. To achieve that goal, an experimental setup have been developed to measure the transmission spectra of the samples under study in different frequency bands (0.220-0.325, 0.843-1.100, and 0.7-1.7 THz depending on the source), and under different experimental conditions (temperature, orientation, applied electric field). Besides this first setup, another bench have been developed to measure the DC and pulsed current-voltage characteristics at low temperatures. The results show a significant enhancement on the transmission spectra as the applied electric field is increased.

Magnetic nanoparticles have great potential in the biomedical and diagnostics field. Due to their small size, the particles have a high surface-to-volume ratio which enables for biofunctionalisation with different molecular probes. This makes itpossible to target them against a wide variety of biomarkers. In this project, the aim was to develop a magnetic nanoparticle-based DNA detection method with respectto sensitivity by employing circle-to-circle amplification, which is an extension of rolling circle amplification, in order to increase the assay sensitivity. The method provides high specificity due to the use of padlock probes for amplification. The project included testing and optimising the protocol used for DNA amplification and detection with a synthetic target, which involved testing different padlock probes, incubation times and incubation temperatures. Lastly, the method was tested on a biological target. It has recently been shown that specific aggregation occurs between magnetic nanoparticles and DNA, which enables for a visual readout strategy sincethe aggregates are visible to the naked eye. Initial testing of the method yielded asensitivity of about 100 attomoles. The achieved sensitivity after the optimisation work was 1 attomole of both synthetic and biological DNA targets. This is an improvement compared to the 400 attomoles that has previously been reported with one round of rolling circle amplification. The results can be used in further development of the naked-eye DNA detection method towards the realisation of a commercially attractive bioanalytical device.

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

In this dissertation, we present analytical and experimental investigations of the electrically actuated micro resonators, when using multi-frequency and/or multi-mode excitation, combined with partial electrodes. We aim to understand their interesting frequency performance and use it to improve the sensing and actuation in microelectromechanical systems (MEMS) and explore their potential applications, such as amplification, gas sensing, magnetometer, multi-physical sensors, and digital-to-analog converters. In the first part, we propose a method of the multi-mode excitation (MME). The concept of the multi-mode excitation is demonstrated by utilizing the superposition principle of two vibration modes in the same phase. To fully understand the difference between the single source excitation (SSE) and multi-mode excitation, we derive the dynamic equations of motions of the electrically-actuated micro cantilever beam and clamped-clamped beam actuated by single/multi-mode excitation. Then, we analytically solve the equations based on the procedure of the Galerkin method with five modes. The simulated results indicate that the MME is clearly superior to that of the SSE, as it can amplify the amplitude performance and signal-to-noise ratio of micro resonators. In the second part, we aim to experimentally prove the concept of the multi-mode excitation and explore its use for gas sensing applications. First, we experimentally investigate the performance of MEMS resonators by single source excitation and multi-mode excitation. We prove the feasibility of the MME approach in enhancing the higher-order mode response for both cantilever and clamped-clamped beams, respectively. We prove that the multi-mode excitation approach provides a better way to activate the higher-order modes with an improved amplitude under a small actuation compared to using a single-source excitation. We then show an improved performance for gas detection. In the third and fourth parts, we propose a technique based on multi-mode excitation for simultaneous sensing for two physical parameters: magnetic field and gas concentration. We respectively investigate a single out-of-plane/in-plane device for in-plane/out-of-plane magnetic field and gas concentration sensing based on tracking the first two vibration modes of a heated buckled micro-beam. We found that operating the resonator at the post-buckling regime, the magnetometer is gas-independent since the first antisymmetric mode (f2) is unaffected by the thermal axial load. Based on it, we utilized the first resonance frequency f1 to detect the gas based on the cooling/heating effects while the second resonance frequency f2 to sense the in-plane/out-of-plane magnetic field. The obtained results demonstrated the sensor acts as a magnetometer and gas sensor, showing good sensitivity, linearity and repeatability. Thus, this technique provides a good candidate for multi-environment monitoring applications. In the last part, we aim to investigate the effects of partial electrodes actuation on the micro resonator and explore its application on the digital-to-analog converter. We analytically and experimentally present modeling, investigation, validation, and optimization of the MEMS resonator-based 3-bit digital to analog converter (DAC) consisting of an in-plane clamped-clamped beam actuated by partial electrodes with different air gaps. The results suggest that the proposed modeling, simulations, and optimization analysis could be successfully implemented in the design of the DAC under various digital combinations. The rich nonlinear behavior with low energy consumption could provide some high potential applications in IoT, such as logic, computation, sensing, and actuation.

Immunoassays are widely used for detection of antigens in a wide range of applications including assays in pharmaceutical development. Immunoassays are continuously improved in many aspects including automatization, miniaturization and extending the dynamic range. The need to measure low abundance molecules are challenging and the need to improve the sensitivity is desired. The Gyrolab technology is a miniaturized immunoassay performed in an automated system covering a broad concentration range. In order to  extend the sensitivity, the technology is combined with Binding Oligo Ladder Detection (BOLD) amplification. The technology behind BOLD or Exazym ® utilizes a DNA primer, a polymerase, and a template (RNA) to generate a ladder-like modified DNA strand. Antibodies with affinity for the polymerized DNA:RNA hybrid strand (duplex) conjugated with reporter molecules are introduced to the system, resulting in an increased number of signal-generating molecules associated with each bound analyte molecule. In this thesis, the development of an ultra-sensitive immunoassay is pursued by applying Exazym ® add-on reagents to the Gyrolab platform, comparing performance with the standard Gyrolab sandwich assay and other commercially available high-performing TNF-α assays. The work includes characterization of a wide range of reaction variables involved in the BOLD signal amplification process including hybridization, polymerization, and detection of a synthetic oligonucleotide. The breakthrough involves the introduction of Allophycocyanin (APC) as a fluorescent conjugate, significantly improving sensitivity and signal-to-noise ratios. The BOLD amplified sensitivity for the TNF-α assay approaches levels seen in ultra-sensitive biomarker assays like Erenna ® and Simoa®. Exazym® technology on the Gyrolab platform allows highly sensitive biomarker assays with minimal sample volume and a 1–2-hour run-time. The study marks substantial progress in achieving ultra-sensitive biomarker assays on the Gyrolab platform through BOLD signal amplification. The use of APC-conjugated detection reagents holds promise for future optimization studies.