Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology.  This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA.  In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap.

The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia. Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function. In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

Topography effects, the modification of seismic motion by topographic features, have been long recognized to play a key role in elevating seismic risk. Site response, the modification of ground motion by near surface soft soils, has been also shown to strongly affect the amplitude, frequency and duration of seismic motion. Both topography effects and 1-D site response have been extensively studied through field observations, small-scale and field experiments, analytical models and numerical simulations, but each one has been studied independently of the other: studies on topography effects are based on the assumption of a homogeneous elastic halfspace, while 1-D site response studies are almost exclusively formulated for flat earth surface conditions. This thesis investigates the interaction between topographic and soil amplification, focusing on strong ground motions that frequently trigger nonlinear soil response. Recently, a series of centrifuge experiments tested the seismic response of single slopes of various inclination angles at the NEES@UCDavis facility, to investigate the effects of nonlinear soil response on topographic amplification. As part of this collaborative effort, we extended the search space of these experiments using finite element simulations. We first used simulations to determine whether the centrifuge experimental results were representative of free-field conditions. We specifically investigated whether wave reflections caused by the laminar box interfered with mode conversion and wave scattering that govern topographic amplification; and whether this interference was significant enough to qualitatively alter the observed amplification compared to free-field conditions. We found that the laminar box boundaries caused spurious reflections that affected the response near the boundaries; however its effect to the crest-to-free field spectral ratio was found to be insignificant. Most importantly though, we found that the baseplate was instrumental in trapping and amplifying waves scattered and diffracted by the slope, and that in absence of those reflections, topographic amplification would have been negligible. We then used box- and baseplate-free numerical models to study the coupling between topography effects and soil amplification in free-field conditions. Our results showed that the complex wavefield that characterizes the response of topographic features with non-homogeneous soil cannot be predicted by the superposition of topography effects and site response, as is the widespread assumption of engineering and seismological models. We also found that the coupling of soil and topographic amplification occurs both for weak and strong motions, and for pressure-dependent media (Nevada sand), nonlinear soil response further aggravates topographic amplification; we attributed this phenomenon to the reduction of apparent velocity that the low velocity layers suffer during strong ground motion, which intensifies the impedance contrast and accentuates the energy trapping and reverberations in the low strength surficial layers. We finally highlighted the catalytic effects that soil stratigraphy can have in topographic amplification through a case study from the 2010 Haiti Earthquake. Results presented in this thesis imply that topography effects vary significantly with soil stratigraphy, and the two phenomena should be accounted for as a coupled process in seismic code provisions and seismological ground motion predictive models.

Surface plasmon-polaritons are optical surface waves formed through the interaction of photons with free electrons at the surface of metals. They offer interesting applications in a broad range of scientific fields such as physics, chemistry, biology, and material science. However, many of such applications face limitations imposed by the high propagation losses of these waves at visible and near-infrared wavelengths, which result mainly from power dissipation in the metal. In principle, the propagation losses of surface plasmon-polaritons can be compensated through optical amplification. The objective of this thesis is to provide deeper insights on the physics of surface plasmon-polariton amplification and spontaneous emission in surface plasmon-polariton amplifiers through theoretical and experimental vehicles applied (but not necessarily restricted) to a particular plasmonic mode termed long-range surface plasmon-polariton. On the theoretical side, the objective is approached by developing a realistic theoretical model to describe the small-signal amplification of surface plasmon-polaritons in planar structures incorporating dipolar gain media such as organic dye molecules, rare-earth ions, and quantum dots. This model takes into account the inhomogeneous gain distribution formed near the metal surface due to a non-uniform excitation of dipoles and due to a position-dependent excited-state dipole lifetime that results from near-field interactions between the excited dipoles and the metal. Also, a theoretical model to describe the amplified spontaneous emission of surface plasmon-polaritons supported by planar metallic structures is developed. This model takes into account the different energy decay channels into which an exited dipole located in the vicinity of the metal can relax. The validity of this model is confirmed through experimentation. On the experimental side, the objective is approached by providing a direct experimental demonstration of complete loss compensation in a plasmonic waveguide. The experiments are conducted using the long-range surface plasmon-polariton supported by a symmetric thin gold waveguide incorporating optically pumped organic dye molecules in solution as the gain medium. Also, an experimental study of spontaneous emission in a long-range surface plasmon-polariton amplifier is presented. It is shown that this amplifier benefits from a low spontaneous emission into the amplified mode, which leads to an optical amplifier with low noise characteristics. The experimental setup and techniques are explained in detail.

Detonation waves are supersonic combustion waves that have a multi-shock front structure followed by a spatially non-uniform reaction zone. During propagation, a de-coupled shock-flame complex is periodically re-initiated into an overdriven detonation following a transient Mach reflection process. Past researchers have identified mechanisms that can increase combustion rates and cause localized hot spot re-ignition behind the Mach shock. But due to the small length scales and stochastic behaviour of detonation waves, the important mechanisms that can lead to re-initiation into a detonation requires further clarification. If a detonation is allowed to diffract behind an obstacle, it can quench to form a de-coupled shock-flame complex and if allowed to form a Mach reflection, re-initiation of a detonation can occur. The use of this approach permits the study of re-initiation mechanisms reproducibly with relatively large length scales. The objective of this study is to experimentally elucidate the key mechanisms that can increase chemical reaction rates and sequentially lead to re-initiation of a de-coupled shock-flame complex into an overdriven detonation wave following a Mach reflection. All experiments were carried out in a thin rectangular channel using a stoichiometric mixture of oxy-methane. Three different types of obstacles were used - a half-cylinder, a roughness plate along with the half-cylinder and a full-cylinder. Schlieren visualization was achieved by using a Z-configuration setup, a high speed camera and a high intensity light source. Results indicate that forward jetting of the slip line behind the Mach stem can potentially increase combustion rates by entraining hot burned gas into unburned gas. Following ignition and jet entrainment, a detonation wave first appears along the Mach stem. The transverse wave can form a detonation wave due to rapid combustion of unburned gas which may be attributed to shock interaction with the unburned gas. Alternatively, the Kelvin-Helmholtz instability can produce vortices along the slipline that may lead to mixing between burned-unburned gases and potentially increase combustion rates near the transverse wave. However, the mechanism(s) that causes the transverse wave to re-initiate into a detonation wave remains to be satisfactorily resolved.

It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution. In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level. These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed. The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population. / Människokroppen är uppbyggd av miljarder celler och nästan alla innehåller samma arvsmassa. Trots detta finns det många olika celler med olika funktioner vilket är en följd av vilken del av arvsmassan som cellerna använder, dvs vilka RNA-molekyler som finns i varje cell. Den snabba utvecklingen av sekvenseringstekniker har gjort det möjligt att studera när, var och hur varje RNA-molekyl är uttryckt och att få en djupare förståelse för hur människans celler fungerar. Arbetet som presenteras i denna avhandling fokuserar på analys av RNA-molekyler i människans celler. I artikel I beskriver vi en automatiserad metod för att förbereda cellprov för RNA-sekvensering. Det automatiserade protokollet jämfördes med det manuella protokollet, och vi visade att det automatiserade protokollet överträffade det manuella när det gällde provkapacitet samtidigt som en höga reproducerbarheten behölls. I artikel II undersökte vi effekterna som RNA-molekyler från en del av cellen (cellkärnan) har på den totala mängden uttryckta RNA-molekyler. Vi jämförde RNA från hela cellen och från en del av cellen (cytoplasman) och visade att RNA-molekyler med långa och strukturerade 3'- och 5'-otranslaterade regioner och RNA-molekyler med långa proteinkodande sekvenser tenderade att hållas kvar i cellkärnan till en högre grad. Detta resulterade i en ökad komplexitet av RNA-molekylerna i hela cellen, medan vi i cytoplasma-fraktionen lättare kunde hitta de korta och svagt uttryckta RNA-molekyler. I Artikel III och IV studerar vi RNA-molekyler i människans skelettmuskler. I artikel III visar vi att andelen RNA-molekyler uttryckta i skelettmuskler är väldigt lika mellan muskler och mellan olika personer, men att ett stort antal RNA-molekyler var uttryckta i olika nivåer hos kvinnor och män. Artikel IV beskriver RNA-nivåer som svar på upprepade perioder av uthållighetsträning. Artikel V beskriver en metod för att studera ett fåtal utvalda RNA-molekyler. Vi valde RNA-molekyler vars uttryck är viktigt vid analys av bröstcancerceller, och optimerade metoden för analys av enskilda celler. Vi analyserade cancerceller från blodprov och använde metoden för att titta på RNA-nivåer i enskilda celler från en grupp av celler och visade på skillnader i RNA-nivåer inom gruppen. / <p>QC 20150115</p>

Chromosomal rearrangements play a major role in the evolution of eukaryotic genomes. Genomic aberrations are also a hallmark of many tumors and are associated with a number of hereditary diseases in humans. The presence of repetitive sequences that can adopt non-canonical DNA structures is one of the factors which can predispose chromosomal regions where they reside to instability. Palindromic sequences (inverted repeats with or without a unique sequence between them) that can adopt hairpin or cruciform structures are frequently found in regions that are prone for gross chromosomal rearrangements (GCRs) in somatic and germ cells in different organisms. Direct physical evidence was obtained that double-strand breaks (DSBs) occur at the location of long inverted repeats, a triggering event for the genomic instability. However, the mechanisms by which palindromic sequences lead to chromosomal fragility are largely unknown. The overall goal of this research is to elucidate the mechanisms of DSB and GCR generation by palindromic sequences in yeast, Saccharomyces cerevisiae.

Els dos principals objectius d'aquesta tesis són: (1) la realització d'un estudi global, (2) anàlisi i caracterització d'alteracions recurrents, en els canvis de metilació genòmica associats al càncer de còlon. Amb la finalitat d'assolir aquests objectius, vàrem desenvolupar una nova tècnica d'anàlisi dels canvis de metilació. Amplification of InterMethylated Sites (AIMS). A partir de la descripció d'aquesta tècnica, la tesis consta de dos grups de resultats, obtinguts a partir de la mateixa tècnica AIMS, i clarament diferenciats. Un primer bloc de resultats on realitzem dos estudis globals (objectiu número 1), i un segon bloc format per dos estudis específics (objectiu número 2) . En el primer treball global es va analitzar la contribució dels guanys i les pèrdues de la metilació genòmica en el procés tumoral. La principal conclusió d'aquest treball va ser el paper independent que juguen els dos tipus de canvi, tant a nivell de característiques tumorals (fisiològiques i genètiques), com de valor pronòstic i contribució al llarg de la progressió tumoral. En el segon estudi global, ens vàrem centrar més amb les pèrdues de metilació genòmica. En aquest, mostrem l'estreta associació entre aquestes pèrdues amb el grau de dany genòmic i mal pronòstic. El segon bloc de resultats ve constituït per els estudis específics. En un primer treball, descrivim el silenciament epigenètic del gen sintasa de prostaciclines (PTGIS). Aquest silenciament, es dona en una freqüència elevada de tumors i presenta una associació estadísticament significativa amb el grau d'aneuploidia del tumor. Finalment, un segon treball específic on mostrem el silenciament epigenètic de tota una banda citogenètica, concretament 2q14.2. En aquest silenciament no tant sols s'hi troba implicada la metilació genòmica, sinó que també descrivim un efecte general de silenciament a través de la modificació post-transcripcional de les histones. / The main focus of this thesis is to better understand the role of genomic methylation changes in colorectal cancer. We approached it at two different levels: (1) global assessment and (2) analysis of specific recurrent changes. A new technique called Amplification of InterMethylated Sites (AIMS) was developed to obtain information at both levels. At global level we report the relative contribution of losses and gains of DNA methylation on the tumour progression. DNA methylation signatures associate with different tumour features, including physiological, genetic and clinical characteristics. Furthermore, we demonstrate that global genomic demethylation correlates with cumulated genomic damage and poor prognosis. At specific level we show the epigenetic silencing of the prostacyclin synthase gene (PTGIS) in 43% of colorectal cancers. PTGIS inactivation is associated with the aneuploid status of the tumour, suggesting a possible role of this gene in the maintenance of the genomic integrity. Finally, we have detected a new type of epigenetic alteration affecting a large proportion of colorectal cancers. It consists in a long range epigenetic silencing due DNA methylation and Histone modification changes and affects an entire cytogenetic band (2q14.2). All the genes and transcripts present in this region showed downregulation independently of the promoter methylation status suggesting that the regional condition prevails over the local status.

The polymerase chain reaction (PCR) is an extremely powerful tool for viral detection and screening because it can detect specific infectious agents with great sensitivity and specificity. It works by exponentially amplifying a target viral DNA sequence to high enough concentrations through the use of specific reagents and thermal cycling. It has surpassed culture based methods as the gold standard for viral detection because of the increased speed and sensitivity. Microfluidic approaches to PCR have focused on decreasing the time to thermally cycle, the volumes used for reactions, and they have also added upstream and downstream processes that are of benefit for on-chip viral detection. While these improvements have made great strides over commercially available products in terms of speed, cost, and integration, a major limitation that has yet to be explored is the throughput associated with running PCR. Since each PCR reaction relies on primers with a unique annealing temperature to detect specific viral DNA, only a single virus can be screened for at a time. The device presented here uses two infrared laser diodes that are driven identically by the same laser driver to independently thermally cycle two chambers on the same microfluidic chip. Different temperatures are achieved in the two chambers by modulating the radiation reaching one of those chambers with an optical shutter. Closed loop temperature feedback in both chambers is done with a Labview program and thermocouples embedded in the polymer chip. This allows for accurate temperature measurement without inhibiting the reaction. To demonstrate the capabilities of this device, two different reactions were simultaneously amplified successfully on the same device that have annealing temperatures that differ by 15°C.