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Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity. The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding. Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments. / <p>QC 20121126</p>

Increasing evidence supports the use of physical activity for modifying brain activity and overall neurological health (Hillman et al, 2008). Specifically, aerobic exercise appears to improve cognitive efficacy with regards to decisional oddball tasks shown through the P300, whose amplitude and latency is augmented (Magnié et al., 2000). Furthermore, the effects of an acute bout of aerobic exercise on cardiovascular function are well established and are sustained following exercise cessation. Based on these findings, we proposed that (1) an acute bout of exercise may modulate movement-related cortical excitability within motor areas and (2) that transient effects would be sustained as long as heart rate (HR) remained elevated. Subjects (n=23) were placed in a soundproof booth and instructed to perform a self-paced unimanual ballistic wrist extension every 3-6 seconds of the right wrist while holding a moveable handle. The motion involved a brisk contraction followed by relaxation and positional reset, collected continuously for approximately 8 minutes. Electroencephalography was used to measure movement-related cortical activity of the Bereitschaftspotential (BP) time-locked to onset of muscle activity associated with movement. The BP is a slow negative self-paced movement related cortical potential that precedes movement by approximately 1500ms. Current work commonly separates the BP into 3 main components early, late, and re-afferent Potentials. The early BP is representative of motor preparation of supplementary motor area (SMA) activity while the late component is representative of motor execution from primary motor cortex (M1). Early and late components are often distinguished by a a characteristic change in slope; where the early BP is a slow negative rise and the late components a steeper negative deflection beginning approximately 500ms prior to movement onset. Broken down further the late component consists of a portion of negative slope before giving rise to a peak approximately 100ms after movement onset known as the motor potential (MP). Following baseline measures, subjects performed 20 minutes of aerobic exercise at a moderate intensity (70% of age-predicted maximum heart rate) on a recumbent cycle ergometer. After the cessation of exercise, BP measures were recorded at two time points: immediately post-exercise (Post) and following a return to iv baseline HR (Post[Rest]) and two additional measures separated by 15 minutes each (Post[Rest2] and Post[Rest3]) which was, on average, 45 minutes after the cessation of exercise. Electromyography (EMG) was employed over the extensor carpi radialis muscle belly to describe muscle burst activity and onset characteristics. Results determined that Early but not Late BP was influenced by aerobic exercise. This early movement related cortical adaptation is indicative of enhanced processing within supplementary motor area. Moreover, this effect was sustained for up to an hour and 15 minutes following exercise cessation. This data is suggestive that aerobic exercise influences on motor related cortical excitability is not driven by an aerobic exercise effect and is more indicative of a delayed neurotransmitter effect.

Single crystal surfaces can be used as substitutes or models for the actual reactive surfaces of heterogeneous catalysts. The advantage in using such models is that the reaction of interest can be isolated on the particular face of the crystal that is thought to be catalytically active. A variety of ordered chlorine covered Ti surfaces were prepared which should be useful as model surfaces for studying Ziegler-Natta reactions. These include a chlorine covered Ti(100) surface on which a 4x6 LEED pattern was observed and a chlorine covered Ti(001) surface, on which a chlorine coincidence lattice was formed. The reactivity of clean and partially chlorine covered Ti(001) and (100) surfaces toward ethylene was also studied. During the investigation of ethylene uptake an epitaxial layer of TiC(lll) was produced on the clean Ti(001) surface. Ethylene uptake curves and carbon segregation to the chlorine covered crystal face are reported for various fractional chlorine coverages. Sulfur segregation from the bulk, to the surface of a Ti single crystal was studied and ordered LEED patterns recorded on ,both the Ti(001) and Ti(100) surfaces. The sulfur covered surfaces were in turn used in additional experiments designed to investigate the uptake of chlorine and the thermal desorption of chlorine from a sulfur covered surface. / Graduation date: 1991

Biological sample analysis is a costly and time-consuming process. It involves highly trained technicians operating large and expensive instruments in a temperature and dust controlled environment. In the world of rising healthcare cost, the drive towards a more cost-effective solution calls for a point-of-care device that performs accurate analyses of human blood samples. To achieve this goal, today's bulky laboratory instruments need to be scaled down and integrated on a single microchip of only a few square centimeters or millimeters in size. Dielectrophoresis (DEP), a phenomenon where small particles such as human blood cells are manipulated by non-uniform electric fields, stands to feature prominently in the point-of-care device. An original device that enhances DEP effect through novel geometry of the electrodes is presented. When activated with two inverting sinusoidal waveforms, the novel-shaped electrodes generate horizontal bands of increasing electric fields on the surface of the microchip. With these bands of electric fields, particles can be manipulated to form a straight horizontal line at a predictable location. Experimental results showing the collection, separation, and transportation of mammalian cells are presented. A strategy for simultaneous processing of two or more types of particles is also demonstrated. With capabilities for an accurate position control and an increased throughput by parallel processing, the novel microchip device delivers substantial improvements over the existing DEP designs. The research presented here explores the effects of novel electrode geometries in cell manipulations and contributes to the overall progress of an automated blood analysis system.

In October 2009, United States Secretary of Education Arne Duncan declared in a speech to Columbia University’s Teacher’s College that many university teacher preparation programs are outdated and must undergo major reform in order to produce high quality teachers needed to improve academic achievement for all students (U.S. Department of Education, 2009). Duncan stated that “America’s university-based teacher preparation programs need revolutionary change – not evolutionary tinkering” (U.S. Department of Education, 2009, p.2). To improve student success in the classroom, policy makers must understand the key role well-trained teachers play in achieving this goal (Boyd, Lankford, Clothfelter, Ladd & Vigdor, 2004; Loeb, Rockoff, & Wyckoff, 2007; Provasnik & Young, 2003; Rice, 2003; Rivers & Sanders, 2002). This study examined the specific aspects of an English teacher preparation program that beginning teachers implement and rely on in their classrooms on a consistent basis. In addition, this study examines how administrators/department chairs view the pedagogical competence of graduates from the English teacher preparation program. The research questions that guided this study are: (1) How do beginning teachers perceive their preparation for teaching in the urban English Language Arts classroom? (2) How do school administrators perceive the teaching ability of graduates? The participants were graduates of Southern Urban University’s English Education Master’s level program from 2005 – 2008. Data sources included Beginning Teacher Questionnaires, Administrator/Department Chair Questionnaires, in-depth phenomenological interviews with select teachers, observations of select teachers’ practice, “card sort” activity/interview, teacher artifacts and photographs. Data were analyzed inductively using the constant comparative method to determine categories and themes (Glaser & Strauss, 1967). Trustworthiness was established through research methods that confirm credibility, transferability, dependability, and confirmability (Lincoln & Guba, 1985). This study provides insight into how to better educate high quality teachers through the examination of an English teacher preparation programs’ daily effect and impact on their graduates and an examination of school administrators’/department chairs perception of these graduates’ performance in the classroom.

Biological sample analysis is a costly and time-consuming process. It involves highly trained technicians operating large and expensive instruments in a temperature and dust controlled environment. In the world of rising healthcare cost, the drive towards a more cost-effective solution calls for a point-of-care device that performs accurate analyses of human blood samples. To achieve this goal, today's bulky laboratory instruments need to be scaled down and integrated on a single microchip of only a few square centimeters or millimeters in size. Dielectrophoresis (DEP), a phenomenon where small particles such as human blood cells are manipulated by non-uniform electric fields, stands to feature prominently in the point-of-care device. An original device that enhances DEP effect through novel geometry of the electrodes is presented. When activated with two inverting sinusoidal waveforms, the novel-shaped electrodes generate horizontal bands of increasing electric fields on the surface of the microchip. With these bands of electric fields, particles can be manipulated to form a straight horizontal line at a predictable location. Experimental results showing the collection, separation, and transportation of mammalian cells are presented. A strategy for simultaneous processing of two or more types of particles is also demonstrated. With capabilities for an accurate position control and an increased throughput by parallel processing, the novel microchip device delivers substantial improvements over the existing DEP designs. The research presented here explores the effects of novel electrode geometries in cell manipulations and contributes to the overall progress of an automated blood analysis system.

The work presented in this thesis aims to further the understanding and implementation of the Baculovirus Expression Vector System (BEVS) for varied uses such as protein production and viral vector production. To this end, three projects have been presented, two of which deal with methods to quantify baculovirus titres and the last deals with tracking baculovirus transcripts in infected insect cells. The first project examined assumption-free analysis as a method for data analysis of Real Time PCR data in order to enable direct comparison of baculovirus titres between samples, without the need for a traditional standard curve. It concluded that assumption-free analysis was well suited for this purpose and fold differences of baculovirus titres of different samples obtained using this method corresponded to real differences in sample titres. The second project aimed to develop a cheap and reliable method for sample preparation for Real Time PCR which would remove the need for the use of commercially available extraction kits. Samples were subjected to various combinations of Triton X-100 at different concentrations and different numbers of freeze/thaw cycles in order to determine the combination which would provide the best baculovirus genome exposure. One of these combinations was found to be at least as good as commercially available kits in reliably extracting baculovirus DNA and providing baculovirus titres that are at least as accurate. The third project was a preliminary study examining the effects of multiplicity of infection on the levels of baculovirus Gp-64 transcript in insect cell culture. The study concludes that at high multiplicities of infection, there seems to be no increase in baculovirus transcripts when the multiplicity of infection is further increased. This study served to allow for familiarization with tracking transcript levels, and the principles and techniques demonstrated here will form the basis for an exhaustive future study on the same subject.

Since the implementation of Directive 2004/48 EC on the Enforcement of Intellectual Property Rights in Sweden there is today a possibility for pharmaceutical companies holding a patent to issue interim injunctions against a generic company on the grounds of an attempt or preparation to a pharmaceutical patent infringement. It has been shown that one of the earliest steps in which a generic company may infringe a pharmaceutical patent is to offer the generic drugs onto the market. However, since the implementation of the attempt and preparation rule in the Swedish Patent Act questions have been raised as to in what stage interim injunctions may be issued against an attempt or preparation to offer generic drugs onto the market made by generic companies. This thesis has therefore intent to investigate at what stage interim injunctions may be applied for against a generic company on the grounds of an attempt or preparation to offer generic drugs. In the thesis the writer argues that in order for an attempt or preparation to offer generic drugs to exist an overall assessment must be made of the particular situation and that there is no specific principles to follow in order to determine either an attempt or a preparation to offer generic drugs. However, some guidance might be brought from Danish case law in order to establish a preparation to offer generic drugs in Sweden.

While a vast amount of research in marketing has examined how information prior to purchase helps consumers to make purchase decisions, relatively little work has considered how marketers can increase the value consumers derive from subsequent experiences using this information. This dissertation develops a construct called "experiential preparation" that describes how consumers can increase the hedonic benefit of their experiences. This dissertation defines "experiential preparation" as any mechanism that allows consumers to familiarize themselves with upcoming experiences in advance of consumption, while the "preparation effect" refers to the increase in liking for an event due to experiential preparation.In a series of ten experimental studies this dissertation demonstrates that experiential preparation increases satisfaction, particularly where the respondent is in a positive mood. It also identifies the primary mechanism through which experiential preparation works, showing that increased satisfaction is fully mediated by fluency. These effects occurred across a range of experiences and modes of preparation. In all the studies, participants viewed feature-length and short, films and read short stories. Participants who engaged in experiential preparation received previews in the form of plot summaries or actual excerpts from the films and stories. In all studies, participants reported their enjoyment for the experiences, and, in several studies additional preference measures were collected. Finally, measures were developed to test for the ways in which fluency mediates and positive moods moderate the preparation effect.This dissertation is organized in three chapters. In Chapter One, experiential preparation and the preparation effect are defined, and background literature is discussed. Chapter Two analyses the results of the ten studies thematically around various mechanisms, some of which have a significant impact on the preparation effect, and some, little impact. Chapter Three presents the studies' results in detail. / Dissertation