Retrovirus gene therapy vectors can deliver therapeutic genes to mammalian cells in a permanent manner by integrating their genome into host chromosomes and therefore provide the potential for long term therapeutic gene expression. Retrovirus integration, however, can be oncogenic. Apart from insertional mutagenesis (IM) genotoxicity may be caused by other factors including DNA damage following infection and integration and epigenetic effects related to incoming viral particles. Thus, using retrovirus and lentivirus infected murine tumour tissue and infected cell lines in vitro this thesis was directed at investigating whether virus infection and integration could cause genotoxicity by alternative route(s) other than IM. Using clonally derived liver tumours that developed in mice, and normal liver and kidney tissues, following EIAV and HIV delivery in utero, comparative genome hybridisation methodology was used to examine for copy number variation. This showed amplification and deletions only in EIAV derived tumours. Real time Q-PCR analysis was then used to measure gene expression changes relating to genes contained within or near to amplifications observed in two tumours of individual mice. The STRING database was then used to find networks linking genes with differential expression profiles and genes in one of these tumours identified with provirus insertions that were also differentially expressed. These data provided preliminary data implicating a role for LV in Hepatocellular carcinoma (HCC). DNA damage is known to cause chromosomal instability that can lead to tumour development. The relationship between double strand breaks (DSB) and virus infection was also investigated in-vitro to find alternative routes to genotoxicity other than IM. Cell viability analysis demonstrated cells with a defective DNA damage response (DDR) have decreased cell viability compared with cells with intact DDR when infected with RV or LV vectors. DSB assays showed RV and LV infection to generate foci over a 6 hour period followed by DDR. Where no viral integrase is present, no DDR appears, however, where the vector is used with or without a genome to infect cells, DDR occurs as shown by the presence of 53BP1 foci indicative of DNA damage. The relationship between DNA damage and methylation was also investigated. Global methylation was found elevated in the genomic DNA of LV and RV infected cells and not in control uninfected cells. In contrast, methylation changes were not found in infected cells lacking the NHEJ repair pathway. These data suggest the DNA damage response is linked to genome methylation. The E2F transcription factor plays a key role in regulating expression of genes known to control oncogenesis and cancer, and E2F is regulated by methylation of its related target gene promoters. Taking into account all genes in the human genome the number of genes that bind E2F is 32.77%. However, using microarray to represent genes differentially expressed after infection, 59% of these were E2F targets. Overall, taking the data obtained in this thesis into account it may be suggested that RV and LV infection causes a number of potentially related changes to cells that include DNA damage and repair and methylation changes that could influence E2F that is an important factor involved in oncogenesis. Combining this with IM, attenuated RV and LV currently in use for gene therapy may cause genotoxicity to infected cells and increase the risk of oncogenesis especially where DNA damage is not correctly repaired. Further work is required to show in greater detail the extent of this genotoxicity, possible by whole genome sequencing of treated host genomes or cell transformation assays linked to the genotoxicity assays presented here. Collectively these data show that alternative factors to IM might exist that could act independently or synergistically to IM.

The Hilbert scheme of \(n\) points in the projective plane parameterizes degree \(n\) zero-dimensional subschemes of the projective plane. We examine the dual cones of effective divisors and moving curves on the Hilbert scheme. By studying interpolation, restriction, and stability properties of certain vector bundles on the plane we fully determine these cones for just over three fourths of all values of \(n\). A general Steiner bundle on \(\mathbb{P}^N\) is a vector bundle \(E\) admitting a resolution of the form \(0 \rightarrow \mathcal{O}_{\mathbb{P}^N} (−1)^s {M \atop \rightarrow} \mathcal{O}^{s+r}_{\mathbb{P}^N} \rightarrow E \rightarrow 0\), where the map \(M\) is general. We complete the classification of slopes of semistable Steiner bundles on \(\mathbb{P}^N\) by showing every admissible slope is realized by a bundle which restricts to a balanced bundle on a rational curve. The proof involves a basic question about multiplication of polynomials on \(\mathbb{P}^1\) which is interesting in its own right. / Mathematics

Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited by viral vectors and its possible role in shaping these vaccine-elicited adaptive immune responses remains unclear. Here we show that Ad and Pox vectors display profound intra- and inter-group differences in innate immune cytokine and chemokine elicitation. The CD46-utilizing vectors Ad35, Ad26, and Ad48 induced greater anti-viral and proinflammatory cytokines and chemokines relative to Ad5 in vaccinated rhesus monkeys and stimulated human PBMC. Ad fiber protein, as well as other capsid components, influenced resultant Ad vector innate stimulatory phenotypes. Analysis of human sera from Ad26-vaccinated volunteers showed similar anti-viral and proinflammatory cytokine and chemokine elicitation. Mechanistic analysis of Ad innate immune stimulation showed greater amounts Ad35 and Ad26, and small amounts of Ad5, traffic to the late endosome following infection. Innate immune stimulation by all three was reduced by inhibition of endosomal acidification, Cathepsin B, and Caspase-1, suggesting a common set of innate immune sensors triggered by Ads between 0-6 hours post-infection, in agreement with trafficking data showing Ad vector colocalization in the late endosome at similar time points. These data suggest a model mechanism explaining differences in observed Ad vector innate immune stimulation phenotypes. Similar to results obtained with Ad vectors, analysis of innate cytokine and chemokine responses elicited by Pox vectors ALVAC, MVA, and NYVAC showed that all three were distinct, with the canarypox-based vector ALVAC eliciting a unique potent proinflammatory response. Together these results reveal surprising and pronounced differences in innate immune stimulatory properties of viral vectors. Furthermore, these results could lead to possible strategies for targeted construction of vaccines for desired innate immune phenotypes, and have profound implications on vaccine design against infectious diseases and cancers, as well as gene therapy.

Oversampled digital-to-analog converters typically employ an array of unit elements to drive out the analog signal. Manufacturing defects can create errors due to mismatch between the unit elements, leading to a sharp reduction in the effective dynamic range through the converter. Mismatch noise shaping is an established technique for alleviating these effects, but usually anchors the signal band to a fixed frequency location. In order to extend these advantages to tunable applications, this work explores a series of techniques that allow the suppression band of the mismatch noise shaping function to have an adjustable center frequency. The proposed techniques are implemented in hardware and evaluated according to mismatch shaping performance, latency and hardware complexity. / text

published_or_final_version / Statistics / Master / Master of Philosophy

A search is presented for vector-like quarks using 20.3 fb⁻¹ of proton-proton collisions at a center-of-mass energy of √S = 8 TeV collected with the ATLAS detector in 2012 at the CERN Large Hadron Collider. Vector-like quarks are predicted to exist in many theories beyond the Standard Model of particle physics that attempt to resolve the hierarchy problem. Events are selected containing jets including at least one b-jet, sizable missing transverse momentum, large scalar sum of the jet and lepton transverse momenta, and either three leptons or two leptons with the same electric charge. Standard Model processes rarely produce this final state and production of vector-like quarks would lead to an enhanced rate of such events. The data are interpreted in the context of a variety of models including pair production of vector-like quarks T and B, which have the same electric charge as the corresponding Standard Model quarks and can appear in either SU(2) weak isospin singlets, doublets or triplets. In addition single and pair production of the vector-like quark T₅/₃ which appears in an SU(2) weak isospin doublet or triplet and has electric charge 5/3, is considered. A moderate excess of data above the SM background expectation is observed with a significance of less than two standard deviations. The data are used to set limits at 95% Confidence Level (CL) on the new heavy quark mass for the various VLQ models considered using the CL_S method. The vector-like quarks T and B in the singlet model are excluded at 95% CL below a mass of 0.59 TeV and 0.62 TeV respectively. The T₅/₃ is excluded at 95% CL below a mass of 0.74 TeV when only pair production is considered and below 0.75 TeV when both pair and single production are considered.

Vectors based on lentivirus backbones have revolutionized our ability to transfer genesinto many cell types. Lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes causing vector replication. Both of these features arecommonly used in gene therapy and have been used clinically in individuals sufferingfrom cancer, infections and genetic diseases. It has been discovered that T-cells can be genetically modified to be used as effective weapons against cancer: therefore virus mustbe produced to deliver the gene of interest into the T-cells. In this project, lentiviralvectors have been produced to transfer the gene coding for a chimeric antigen receptor(CAR) which is directed to CD19 on B-cells. The vectors will, hence, be used to generateCD19 retargeted T-cells in purpose to kill CD19 cells such as B-cell lymphoma andleukemia. We have evaluated two production protocols to determine a feasible method toculture these vectors. We have also stimulate T-cells with two different antibodies (anti-CD3 and anti-CD28) and transduced T-cells. Our results demonstrate that theconcentration of virus was higher after prolonged incubation in 4˚C, which can not beexplained. The stimulation demonstrated that bound anti-CD3 was the best stimulator,and moreover the FACS-analysis showed that addition of anti-CD28 gave a highertransduction level. In conclusion, the viral vectors may be kept in 4˚C for two days beforeconcentrating the virus, and bound anti-CD3 is a better choice than soluble anti-CD3 forstimulation of T-cells.

Transposons are DNA segments that can move or transpose themselves to new positions within the genome of an organism. Biologists need to predict preferred insertion sites of transposons to devise strategies in functional genomics and gene therapy studies. It has been found that the deformability property of the local DNA structure of the integration sites, called Vstep, is of significant importance in the target-site selection process. We considered the Vstep profiles of insertion sites and developed predictors based on Artificial Neural Networks (ANN) and Support Vector Machines (SVM). We trained our ANN and SVM predictors with the Sleeping Beauty transposonal data, and used them for identifying preferred individual insertion sites (each 12bp in length) and regions (each 100bp in length). Running a five-fold cross-validation showed that (1) Both ANN and SVM predictors are more successful in recognizing preferred regions than preferred individual sites; (2) Both ANN and SVM predictors have excellent performance in finding the most preferred regions (more than 90% sensitivity and specificity); and (3) The SVM predictor outperforms the ANN predictor in recognizing preferred individual sites and regions. The SVM has 83% sensitivity and 72% specificity in identifying preferred individual insertion sites, and 85% sensitivity and 90% specificity in recognizing preferred insertion regions.

In this expository thesis we describe the Bochner integral for functions taking values in a separable Banach space, and we describe how a number of standard definitions and results in real analysis can be extended for these functions, with an emphasis on Hilbert-space-valued functions. We then present a partial vector-valued version of a classical theorem on singular integrals.

Variable speed drive technology has advanced dramatically in the last 10 years with the advent of new power devices. In this study, a three phase induction motor drive using Insulated Gate Bipolar Transistors (IGBT) at the inverter power stage is introduced to implement speed and position control for the revolving stage in the Frederic Wood Theatre This thesis presents a solution to control a 3-phase induction motor using the Texas Instruments (TI) Digital Signal Processor (DSP) TMS320F2407A. The use of this DSP yields enhanced operations, fewer system components, lower system cost and increased efficiency. The control algorithm is based on the constant volts-per-hertz principle because the exact speed control is not needed. Reflective object sensors which are mounted on concrete frame are used to detect accurate edge position of revolving stage. The sinusoidal voltage waveforms are generated by the DSP using the space vector modulation technique. In order to satisfy some operating conditions for safe and agreeable operation, a look-up table, which is used to give command voltage and speed signals in software, is applied to limit the maximum speed and acceleration of the revolving stage. Meanwhile, a boost voltage signal is added at the low frequency areas to make the motor produce maximum output torque when starting. A test prototype is then built to validate the performance. Several tests are implemented into the IGBT drive to explore the reason for unacceptable oscillations in IGBT’s gate control signals. Improvement methods in hardware layout are suggested for the final design.