Les méthodes de retournement temporel (RT) en micro-ondes sont appliquées à l'amplification d'impulsions et à l'imagerie. Lors d'une première partie, une chambre réverbérante ouverte sur sa face avant permet d'engendrer un champ diffus tout en laissant s'échapper l'énergie afin de focaliser le champ par RT à l'extérieur. La compression spatiotemporelle après RT produit une impulsion de forte amplitude et confère au dispositif un caractère auto-adaptatif en position et en polarisation. La seconde partie traite expérimentalement et théoriquement de la méthode DORT (décomposition de l'opérateur de RT). Le cas d'un cylindre diélectrique est examiné dans le but de retrouver ses paramètres. L'imagerie de deux cibles séparées d'une distance sub-longueur d'onde est alors abordée. Un critère de résolution déterminant le niveau de bruit à partir duquel la résolution des cibles échoue est notamment extrait. La méthode est ensuite appliquée à la localisation de personnes mobiles derrière un mur. La possibilité de suivre un déplacement est illustrée en prenant en compte la propagation à l'intérieur du mur. L'influence du déplacement d'une cible ponctuelle pendant l'acquisition de la matrice de transfert sur les invariants de l'opérateur de RT est aussi examinée. Enfin, une technique d'imagerie passive fondée sur les corrélations du bruit ambiant est expérimentalement mise en évidence en micro-ondes. Suivant une analogie avec le RT, la corrélation de signaux de bruit large bande mène à la fonction de Green entre deux antennes et ainsi à la localisation de cibles. La localisation passive d'une personne est aussi abordée en « bande étroite » grâce à l'émission d'une borne WIFI.

Le développement du laser a ouvert la voix à l'exploration de nouveaux domaines scientifiques et industriels. Les impulsions laser à haute intensité sont un outil unique pour les études d'interaction lumière/matière et leurs applications. Mais elles sont générées par des systèmes laser reposant sur l'utilisation de milieux à gain en verre pompés par des lampes flashes et sont donc intrinsèquement limitées en termes de cadence et d'efficacité. Le développement, au cours de ces dernières années, des lasers semi-conducteurs a attiré l'attention sur une nouvelle classe de lasers, les « laser solides pompés par diodes » (DPSSL). Ils possèdent une grande efficacité et sont des candidats de choix pour les systèmes compacts à haute puissance moyenne requis pour des applications industrielles, mais aussi en tant que sources de pompe à haute puissance pour des lasers ultra-intenses. Les travaux décrits dans cette thèse s'inscrivent dans le cadre du système laser Lucia (1 kilowatt de puissance moyenne), actuellement en construction au «Laboratoire d'Utilisation des Intenses lasers» (LULI) à l'Ecole Polytechnique, France. La génération d'impulsions laser de durée sub-10 nanosecondes avec des énergies allant jusqu'à 100 joules et des taux de répétition de 10 hertz est principalement limitée par l'émission spontanée amplifiée (ASE) et les effets thermiques. L'étude de ces limitations est le thème central de ce travail. Leur impact est discuté dans le cadre d'un premier jalon énergétique fixé vers 10 joules. Le système laser mis au point est présenté en détails depuis l'oscillateur jusqu'à la fin de la chaine d'amplification. Une discussion complète de l'impact de l'ASE et des effets thermiques est complétée par des vérifications expérimentales. Les modèles de simulation informatique développés sont validés puis utilisés pour prédire les performances du système laser qui, lors d'une première activation, à atteint un niveau d'énergie de 7 joules en régime mono-coup et de 6,6 joules pour un taux de répétition de 2 hertz. Les limitations actuelles sont discutées ainsi que les approches envisagées pour des développements futurs.

<p>This thesis report is focused on the growth of Pt/Mg multilayers and the studies of the sputter yield amplification effect in these. The main application is to use the multilayers as X-ray mirrors reflecting an X-ray wavelength of 17 Å. This wavelength is important for astronomical applications in general, and solar imaging applications in particular.</p><p>For periodic X-ray multilayer mirrors only a certain specific wavelength of X-rays can be reflected. What wavelength that is reflected depends on the individual layer thicknesses of the materials that are constituting the multilayer. These thicknesses can be determined using modified Bragg’s law and are approximately a quarter of the wavelength.</p><p>In order to obtain the exact desired layer thickness of each individual layer it is necessary to understand the growth processes and the effects that are going on during deposition of such multilayer mirrors. It has been shown that when depositing multilayers consisting of one very light and one very heavy material, like e.g. Pt and Mg, the deposition rate of the light element is non-linear with deposition time for thin layers. This is because of backscattered energetic neutrals from the heavy target material, which affects the growing film. Furthermore, a sputter yield amplification is present for thin layers when a light element is grown on top of a heavy element, i.e. for Mg on top of Pt.</p><p>Dual DC magnetron sputtering has been used to grow the Pt/Mg multilayers, and the influence of the backscattered energetic neutrals and the sputter yield amplification effect has been studied for Ar and Kr sputtering gases at pressures ranging from 3 up to 9 mTorr. The individual layer thicknesses have been obtained from simulations of hard X-ray reflectivity measurements using the IMD program. The number of backscattered energetic neutrals and their energies at the target have been calculated using the TRIM code.</p><p>Using the results obtained it is now possible to predict and compensate for the non-linear deposition rate of Mg.</p>

<p>BACKGROUND: Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder caused by mutations in the gene coding for microsomal triglyceride transfer protein (MTTP).</p><p>AIM: To characterize the genetic basis of ABL in two unrelated patients.</p><p>RESULTS: In the first patient, the substitution c.1911C>T in exon 12 of the <em>MTTP</em> gene, resulting in the protein substitution p.P552L, was discovered using mutation screening. The parents are heterozygous and the proband is a homozygous carrier of this substitution. Using restriction fragment length polymorphism (RFLP), 100 control subjects were analyzed and none carried the substitution indicating that it is a novel <em>MTTP </em>mutation. Sequencing of the other ABL patient showed that the proband carried a homozygous single base insertion, at position  c.2342IVS16+2-3insT, located at the donor splice-site of intron 16 resulting in skipping of exon 16 and truncation of the protein. The proband's mother is heterozygous for the insertion while the father does not carry the insertion. Multiplex ligation-dependent probe amplification (MLPA) did not identify any deletion encompassing exon 16 in the proband, father or mother. Nonpaternity was excluded using polymorphic markers from several chromosomes. Haplotype analysis using markers spanning chromosome 4 revealed  heterodisomy (two homologous chromosomes) of 4p and the distal part of 4q, and isodisomy (duplication of one chromosome) of 4q12-4q26.</p><p>CONCLUSION: These data show that the cause of ABL in one of the patients is a missense mutation, p.P552L, while the cause of ABL in the other patient is due to uniparental disomy, probably resulting from non-disjunstion in meiosis I.</p>

<p>Among the great challenges in biology are the precise quantification of specific sets of proteins and analyses of their patterns of interaction on a much larger scale than is possible today. </p><p>This thesis presents a novel protein detection technique - proximity ligation - and reports the development and application of a nucleic acid amplification technique, RCA. Proximity ligation converts information about the presence or co-localization of specific proteins to unique sets of nucleic acid sequences. For detection of target proteins or protein complexes the coincident binding by pairs or triplets of specific protein-binding reagents are required. Oligonucleotide-extensions attached to those binding reagents are joined by a DNA ligase and subsequently analyzed by standard molecular genetic techniques. The technique is shown to sensitively detect an assortment of proteins using different types of binders converted to proximity probes, including SELEX aptamers and mono- and polyclonal antibodies. I discuss factors important for using the technique to analyze many proteins simultaneously.</p><p>Quantification of target molecules requires precise amplification and detection. I show how rolling circle amplification, RCA, can be used for precise quantification of circular templates using modified molecular beacons with real-time detection. The combination of proximity-probe templated circularization and RCA results in a sensitive method with high selectivity, capable of visualizing individual immobilized proteins. This technique is used for localized detection of a set of individual proteins and protein complexes at sub-cellular resolution.</p>

<p>Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis.</p><p>The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization.</p><p>Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.</p>

<p>In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. </p><p>The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. </p><p>The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women.</p><p>Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. </p><p>A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.</p>

<p>In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.</p><p>The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.</p>

Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis. The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization. Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.

In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women. Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.