Transgenic organisms are a valuable tool for studying epigenetics, as they provide significant insight into the evolutionary conservation of epigenetic control sequences, the interacting proteins, and the underlying molecular mechanisms. Paramutation is an epigenetic phenomenon in which the epigenetic status and expression level of one allele is heritably altered after pairing with another. At the b1 locus in maize, a control region consisting of seven 853 bp tandem repeats is required for paramutation. To study the conservation of the epigenetic mechanisms underlying maize b1 paramutation, I created transgenic Drosophila carrying the maize b1 control region flanked by FRT sites and adjacent to the Drosophila white reporter gene. The maize b1 tandem repeats caused epigenetic silencing in Drosophila, as white expression consistently increased following repeat removal. A single copy of the tandem repeat sequence was sufficient to cause silencing, and silencing strength increased as the number of repeats increased. Trans interactions, such as pairing-sensitive silencing, were also observed and appear to require a threshold number of b1 tandem repeats, similar to paramutation in maize. Analysis of transcription from the repeats showed that the b1 tandem repeats are transcribed from both strands in Drosophila, as they are in maize. Bidirectional transcription was found to extend to the regions flanking the repeats, and persisted in “repeats-out” transgenes following repeat removal. However, aberrant transcription was lost when a zero-repeat transgene was moved to a new genomic position, suggesting that it may be due to an epigenetic mark that is retained from the previous silenced state. A search for modifiers of b1 repeat-mediated silencing demonstrated that Polycomb group proteins are involved. Together, these results indicate considerable conservation of an epigenetic silencing process between the plant and animal kingdoms. Genomic imprinting is a related epigenetic process in which parent-specific epigenetic states are inherited and maintained in progeny. The conservation of epigenetic mechanisms was further explored via an in-depth review of the molecular mechanisms underlying genomic imprinting in plants, mammals and insects, and identification of potentially imprinted genes in Drosophila by microarray analysis.

Tandem mass spectrometry (MS/MS) is a powerful tool for identifying peptide sequences. In a typical experiment, incorrect peptide identifications may result due to noise contained in the MS/MS spectra and to the low quality of the spectra. Filtering methods are widely used to remove the noise and improve the quality of the spectra before the subsequent spectra identification process. However, existing filtering methods often use features and empirically assigned weights. These weights may not reflect the reality that the contribution (reflected by weight) of each feature may vary from dataset to dataset. Therefore, filtering methods that can adapt to different datasets have the potential to improve peptide identification results. This thesis proposes two adaptive filtering methods; denoising and quality assessment, both of which improve efficiency and effectiveness of peptide identification. First, the denoising approach employs an adaptive method for picking signal peaks that is more suitable for the datasets of interest. By applying the approach to two tandem mass spectra datasets, about 66% of peaks (likely noise peaks) can be removed. The number of peptides identified later by peptide identification on those datasets increased by 14% and 23%, respectively, compared to previous work (Ding et al., 2009a). Second, the quality assessment method estimates the probabilities of spectra being high quality based on quality assessments of the individual features. The probabilities are estimated by solving a constraint optimization problem. Experimental results on two datasets illustrate that searching only the high-quality tandem spectra determined using this method saves about 56% and 62% of database searching time and loses 9% of high-quality spectra. Finally, the thesis suggests future research directions including feature selection and clustering of peptides.

The most important issue at a container terminal is to minimize the ship’s turnaround time which is determined by the productivities of quay cranes (QCs). The tandem lift quay cranes have 33% higher productivities than single lift QCs. However, the tandem lift operations bring new challenges to the vehicle dispatching at terminals and this has become a big issue in the application of tandem lift QCs. The vehicle dispatching at terminals is to enhance the QCs’ productivities by coordinating the QCs’ operation schedules and the vehicles’ delivery schedules. The static version of the problem can be formulated as an MILP model and it is a combinational optimization problem. When the type of QC is tandem lift, the problem becomes more complicated because it requires two vehicles side by side under the QC. Thus, the alignments of vehicles have to be considered by coordinating the delivery schedules between vehicles. On the other hand, because the containers are operated alone by the yard cranes, the vehicles could not be grouped and dispatched in pairs all the time. This dissertation investigates the static and dynamic version of the problem and proposes heuristic methods to solve them. For the static version, Local Sequence Cut (LSC) Algorithm is proposed to tighten the search space by eliminating those feasible but undesirable delivery sequences. The time windows within which the containers should be delivered are estimated through solving sub-problems iteratively. Numerical experiments show the capability of the LSC algorithm to find competitive solutions in substantially reduced CPU time. To deal with the dynamic and stochastic working environment at the terminal, the dissertation proposes an on-line dispatching rule to make real-time dispatching decisions without any information of future events. Compared with the longest idle vehicle rule, the proposed priority rule shortens the makespan by 18% and increases the QCs’ average productivities by 15%. The sensitivity analysis stated that the superiority of the priority rule is more evident when the availability of vehicles is not sufficient compared with the frequency of releasing transportation requests.

No description available.

At the Tandem accelerator in the Ångström laboratory, Uppsala, Rutherford backscattering spectrometry (RBS) is one of the methods used for thin film analysis, providing information on thickness and composition. The films are commonly grown on silicon substrates, whose crystal structure gives rise to channelling effects (a strong angular dependence in the intensity of the signal), which can cause faulty results. For other samples, channelling may also be used to get information on crystal structure and quality. This work demonstrates new functions to the existing software, aiming at minimizing these effects. The new methods have been tested by measurements both in channelling directions and in directions determined by the old method. In comparison with the earlier method the worst-case error is of order 80 %,commonly around 20 %, but it is possible to achieve an error which is not detectable. It is worth to note that the stated errors appear in tests oriented for maximum channelling, where effects without the new methods give an error corresponding to an apparent thin-film thickness almost 18 times that of the actual thickness. / Vid Tandemlabbet i Ångströmlaboratoriet, Uppsala, används bland annat Rutherford backscattering spectrometry (RBS) för att undersöka egenskaper, t.ex tjocklek och sammansättning, hos tunnfilmer som ofta är odlade på kiselsubstrat. Kiselkristallernas struktur ger upphov till kanaliseringseffekter, d.v.s starkt vinkelberoende intensitet, som i detta sammanhang kan ge felaktiga resultat. För andra prover kan kanaliseringseffekter användas för att få information om kristallstruktur och kvalitet. I det här arbetet demonstreras nyskrivna funktioner till befintlig mjukvara med syfte att minimera dessa effekter. De nya funktionerna har testats genom provtagningar i orienteringar som är gynnsamma och icke gynnsamma för kanalisering. Vid jämförelse med tidigare metoder ger de nya metoderna ett fel på i värsta fall ca. 80%, med bättre parametrar sjunker felet till ca 20 % och med rätt val av parametrar försvinner felet jämfört med tidigare metod. Värt att notera är att ovanstående maximala fel uppstår vid test orienterat för maximal kanalisering, där effekterna utan de nya metoderna ger ett fel på uppemot en faktor 18.

Nonribosomal peptides (NRP) are highlighted in pharmacological studies as novel NRPs are often promising substances for new drug development. To effectively discover novel NRPs from microbial fermentations, a crucial step is to identify known NRPs in an early stage and exclude them from further investigation. This so-called dereplication step ensures the scarce resource is only spent on the novel NRPs in the following up experiments. Tandem mass spectrometry has been routinely used for NRP dereplication. However, few bioinformatics tools have been developed to computationally identify NRP compounds from mass spectra, while manual identification is currently the roadblock hindering the throughput of novel NRP discovery. In this thesis, we review the nature of nonribosomal peptides and investigate the challenges in computationally solving the identification problem. After that, iSNAP software is proposed as an automated and high throughput solution for tandem mass spectrometry based NRP identification. The algorithm has been evolved from the traditional database search approach for identifying sequential peptides, to one that is competent at handling complicated NRP structures. It is designed to be capable of identifying mixtures of NRP compounds from LC-MS/MS of complex extract, and also finding structural analogs which differ from an identified known NRP compound with one monomer. Combined with an in-house NRP structural database of 1107 compounds, iSNAP is tested to be an effective tool for mass spectrometry based NRP identification. The software is available as a web service at http://monod.uwaterloo.ca/isnap for the research community.

The factors affecting peptide fragmentation have been extensively studied in the literature in order to better predict the fragment ion spectra of peptides and proteins. While there are countless influences to consider, metal cation binding in the gas-phase is particularly interesting. Herein, a comparison of fragmentation patterns of a model peptide series with various charge carriers (H+, Li+, Na+, K+, and Cu+) will assist in determining the location of the preferred binding site of the metal cation and in assessing differences in the fragmentation pattern as a result of this binding site. An interesting observation from these studies reveals abundant x-type fragment ions occurring from the fragmentation of alkali-metal cationized peptides. As these fragment ions have been observed in previous studies by others but not addressed, the factors affecting the formation of these x-type fragment ions are explored. Additionally, a home-built 193-nm photodissociation tandem time-of-flight mass spectrometer is utilized to study how peptide fragmentation kinetics affect the fragmentation pattern observed. Initially, the fragmentation timescales of various peptides are investigated. Results indicate that longer fragmentation timescales (~10 microseconds) result in an increased number of identified peaks with internal and ammonia loss fragment ions being the most common in comparison to 'prompt' fragmentation timescales (~1 microsecond). Furthermore, b-type fragment ion formation is also favored at longer timescales for the arginine containing peptides investigated. The fragmentation pattern of several proline containing peptides is examined by collision-induced dissociation and 193-nm photodissociation. Unique fragment ions are observed with each occurring at a proline residue. Few differences are detected between CID and 193-nm photodissociation spectra, indicating that the proline residues direct fragmentation rather than the dissociation method. In an effort to improve the performance of the photodissociation tandem TOF instrument, the addition of a second source and a dual-stage reflectron are incorporated. The modifications result in improved mass range, signal-to-noise, and increased fragment ion collection efficiencies. High quality mass spectra are acquired across a range of mass-to-charge ratios from ~600 to 1900. Furthermore, the modifications continue to allow investigation of various fragmentation timescales with the addition of an additional timeframe of ~3 microseconds.

The relationship between distinctive characteristic fluid-flow regimes and the sound radiation generated by them has been investigated, over a range of Reynolds numbers, for various single plates and two-plate arrays in nominally two-dimensional flows. In preliminary experiments, the characteristics of flow over single plates with rectangular cross-section and faired leading edges and over tandem arrays of an upstream plate with rectangular cross-section and faired leading edges and a downstream plate of rectangular cross-section were investigated, together with the sound radiation produced. However, the main investigation has been concentrated on single plates of rectangular cross-section with various chord-to-thickness ratios C and on arrays of two plates of rectangular cross-section in tandem having various chord-to-thickness ratios C₁ and C₂ and a range of gaps (with gap-to-thickness ratios G) between them. The range of Reynolds number based on plate thickness t and free-stream velocity U, Re[subscript]t = Ut/ν (where ν is the kinematic viscosity of fluid) covered in the measurements is 3.2 x 10[superscript]3 ≤ Re[subscript]t 53 x 10[superscript]3. Spectra of velocity fluctuations in the flow and radiated sound have been measured and their characteristic frequencies related. An attempt has been made to measure force fluctuations on surfaces of the plates in order to relate them to flow characteristics and radiated sound power. Mean and fluctuating pressures associated with the force fluctuations on the plates have also been obtained. The lengths of separation bubbles on long rectangular plates have also been determined. In most cases, the measurements have been complemented by flow-visualisation in a water tunnel to provide additional detailed insight into the flow patterns. Three flow regimes have been identified for single plates of rectangular cross-section. In the first regime (1 ≤ C ≤ 3.13), shear layers separated from the leading edges form a vortex street downstream of the plate without reattachment to it. Associated force fluctuations on the plate are the main source of acoustic radiation. In the second regime (3.05 ≤ C ≤ 9.65), the separated shear layers reattach intermittently to the streamwise plate surfaces. Vortex formation in the shear layer is the dominant cause of sound radiation but the effect becomes weaker as C increases. In the third regime (6.52 ≤ C ≤ 68), the separated shear layers form closed leading-edge separation bubbles. Weak vortex shedding, with only a small contribution to the sound radiation, occurs only at the trailing edges of the plate. Bistable behaviour of the flow over a plate, with random switching between the regimes, occurs for C ≈ 3 and 6.52 ≤ C ≤ 9.65. A proposed classification of possible flow regimes for the flow around two plates of rectangular cross-section in tandem has been confirmed experimentally. For small G, the flow in the gap between the plates is isolated from the external flow. When the gap G between the plates is increased to or beyond a critical value (between 2 and 3.5), the shear layers separated from the upstream plate form a von Karman vortex street in the gap before interacting with the downstream plate. Flow and acoustic measurements indicate that this transition is associated with dramatic changes in the flow character. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1320474 / Thesis (Ph.D.) -- University of Adelaide, School of Mechanical Engineering, 2008

Microsatellites are short (1-6bp long) highly polymorphic tandem repeats, found in all genomes analyzed so far. Popular genetic markers for many applications including population genetics, pedigree analysis, genetic mapping and linkage analysis, some microsatellites also can cause a variety of human neurodegenerative diseases and may act as agents of adaptive evolution through the regulation of gene expression. As a consequence of these diverse uses and functions, the mutational and evolutionary dynamics of microsatellite sequences have gained much attention in recent years. Mostly, the focus of studies investigating microsatellite evolution has been to develop more refined evolutionary models for estimating parameters such as genetic distance or linkage disequilibrium. However, there is an incentive in using our understanding of the evolutionary processes that affect these sequences to examine the functional implications of microsatellite evolution. What has emerged from nearly two decades of study are highly complex mutational dynamics, with mutation rates varying across species, loci and alleles, and a multitude of potential influences on these rates, most of which are not yet fully understood. The increasing availability of whole genome sequences has immensely extended the scope for studying microsatellite evolution. For example, where once it was common to examine single loci, it is now possible to examine microsatellites using genome wide approaches. In the first part of my dissertation I discuss approaches and issues associated with detecting microsatellites in genomic data. In Chapter 2 I undertook a meta-analysis of studies investigating the distribution of microsatellites in yeast and showed that studies comparing the distribution of microsatellites in genomic data can be fraught due to the application of different definitions for microsatellites by different investigators. In particular, I found that variation in how investigators choose the repeat unit size of a microsatellite, handle imperfections in the array and especially the choice of minimum array length used, leads to a large divergence in results and can distort the conclusions drawn from such studies, particularly where inter-specific comparisons are being made. In a review of the currently available suite of bioinformatics tools (Chapter 3), I further showed that this bias extends beyond a solely theoretical controversy into a methodological issue because most software tools not only incorporate different definitions for the key parameters used to define microsatellites, but also employ different strategies to search and filter for microsatellites in genomic data. In this chapter I provide an overview of the available tools and a practical guide to help other researchers choose the appropriate tool for their research purpose. In the second part of my thesis, I use the analytical framework developed from the previous chapters to explore the biological significance of microsatellites exploiting the well annotated genome of the model organism Saccharomyces cerevisiae (baker’s yeast). Several studies in different organisms have indicated spatial associations between microsatellites and individual genomic features, such as transposable elements, recombinational hotspots, GC-content or local substitution rate. In Chapter 4, I summarized these studies and tested some of the underlying hypotheses on microsatellite distribution in the yeast genome using Generalized Linear Models (GLM) and wavelet transformation. I found that microsatellite type and distribution within the genome is strongly governed by local sequence composition and negative selection in coding regions, and that microsatellite frequency is inversely correlated with SNP density reflecting the stabilizing effect point mutations have on microsatellites. Microsatellites may also be markers for recent genome modifications, due to their depletion in regions nearby LTR transposons, and elements of potential structural importance, since I found associations with features such as meiotic double strand breaks, regulatory sites and nucleosomes. Microsatellites are subject to local genomic influences, particularly on small (1-2kb) scales. Although, these local scale influences might not be as dominant as other factors on a genome-wide scale they are certainly of importance with respect to individual loci. Analysis of locus conservation across 40 related yeast strains (Chapter 5) showed no bias in the type of microsatellites conserved, only a negative influence of coding sequences, which supports again the idea that microsatellites evolve neutrally. Polymorphism was rare, and despite a positive correlation with array length, there was no relationship with either genomic fraction or repeat size. However, the analysis also revealed a non-random distribution of microsatellites in genes of functionally distinct groups. For example, conserved microsatellites (similar to general microsatellites in yeast) are mostly found in genes associated with the regulation of biological and cellular processes. Polymorphic loci show further an association with the organization and biogenesis of cellular components, morphogenesis, development of anatomical structures and pheromone response, which, is absent for monomorphic loci. Whether this distribution is an indication of functionality or simply neutral mutation (e.g. genetic hitch-hiking) is debatable since most conserved microsatellites, particularly variable loci, are located within genes that show low selective constraints. Overall, microsatellites appear as neutrally evolving sequences, but owing to the sheer number of loci within a single genome, individual loci may well acquire some functionality. More work is definitely needed in this area, particularly experimental studies, such as reporter-gene expression assays, to confirm phenotypic effects.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request