Genome-wide analyses using bead-based microarrays

Microarrays are now an established tool for biological research and have a wide range of applications. In this thesis I investigate the BeadArray microarray technology developed by Illumina. The design of this technology is unique and gives rise to many computational and statistical challenges. However, I show how knowledge from other microarray technologies can be used to our advantage. I describe the beadarray software package, which is now used by researchers around the world. The development of this software was motivated by the fact that Illumina's software (BeadStudio) gives a summarised view of Illumina data and does not gives users any control over several processing steps that were found to be crucial for other microarray technologies. A main feature of beadarray is the ability to access raw data. The advantages of such data include the ability to perform more detailed quality assessment and greater control over the analysis at all stages. The analysis of a control experiment shows that the processing steps used in BeadStudio can be improved. In particular, utilising variances calculated from the raw data can increase the ability to detect genes which have di erent expression levels between samples, a common goal for microarray studies. The data from the control experiment are made available for other researchers to use and validate their own analysis methods. One issue discovered during the analysis of the control experiment was that only half of the intended genes could be reliably measured due to problems in the design of the probes targetting particular genes. By considering a large set of publicly available Illumina arrays, I show how such unreliable measurements can a ect the analysis of Illumina data. I also show how potential problems can be identi ed in advance of an experiment and incorporated into an analysis pipeline

Optimizing Conical Intersections of Solvated Molecules: The Combined Spin-Flip Density Functional Theory/Effective Fragment Potential Method

Solvent effects on a potential energy surface crossing are investigated by optimizing a conical intersection (CI) in solution. To this end, the analytic energy gradient has been derived and implemented for the collinear spin-flip density functional theory (SFDFT) combined with the effective fragment potential (EFP) solvent model. The new method is applied to the azomethane-water cluster and the chromophore of green fluorescent protein in aqueous solution. These applications illustrate not only dramatic changes in the CI geometries but also strong stabilization of the CI in a polar solvent. Furthermore, the CI geometries obtained by the hybrid SFDFT/EFP scheme reproduce those by the full SFDFT, indicating that the SFDFT/EFP method is an efficient and promising approach for understanding nonadiabatic processes in solution

Complex WKB Analysis of a PT Symmetric Eigenvalue Problem

The spectra of a particular class of PT symmetric eigenvalue problems has previously been studied, and found to have an extremely rich structure. In this paper we present an explanation for these spectral properties in terms of quantisation conditions obtained from the complex WKB method. In particular, we consider the relation of the quantisation conditions to the reality and positivity properties of the eigenvalues. The methods are also used to examine further the pattern of eigenvalue degeneracies observed by Dorey et al. in [1,2].Comment: 22 pages, 13 figures. Added references, minor revision

Survey-based pilot study into the chosen therapy and prophylaxis used by UK primary care veterinary surgeons against canine angiostrongylosis

Canine Angiostrongylosis (CA), a gastropod-borne parasitic infection caused by the metastrongyloid nematode Angiostrongylus vasorum, is an important cause of significant morbidity to domestic dogs across the UK as well as in other European countries. This study aimed to ascertain the frequency at which particular drugs were used by primary care practitioners in the UK for therapy against and prophylaxis for CA. Primary care veterinary clinicians were surveyed using an online questionnaire and face-to-face or telephone interviews. Eighty-six veterinary surgeons responded. The majority of practices (n = 52) included lungworm in their standard anthelmintic protocols; moxidectin was the most common drug used for prophylaxis (n = 71). Fenbendazole was the most frequently selected drug, by 45% of vets, for treatment of confirmed cases of CA despite it being unlicensed for this purpose in the UK and the absence of a clear treatment protocol. The results of this pilot study provide an initial insight into the approach taken by primary care practitioners in their approach to CA. This provides an important starting point for future studies investigating the decision-making for CA amongst UK veterinary surgeons, particularly to clarify whether in a larger cohort an unlicensed drug remains the treatment of choice. The absence of a clear protocol for fenbendazole means that treatment of dogs affected by CA may be suboptimal, increasing the risk of morbidity and mortality

Telomere length and common disease: study design and analytical challenges.

Telomeres, the repetitive sequences that protect the ends of chromosomes, help to maintain genomic integrity and are of key importance to human health. The aim here is to give an overview of the evidence for the importance of telomere length (TL) to the risk of common disease, considering the strengths and weaknesses of different epidemiological study designs. Methods for measuring TL are described, all of which are subject to considerable measurement error. TL declines with age and varies in relation to factors such as smoking and obesity. It is also highly heritable (estimated heritability of ~40 to 50%), and genome-wide studies have identified a number of associated genetic variants. Epidemiological studies have shown shorter TL to be associated with risk of a number of common diseases, including cardiovascular disease and some cancers. The relationship with cancer appears complex, in that longer telomeres are associated with higher risk of some cancers. Prospective studies of the relationship between TL and disease, where TL is measured before diagnosis, have numerous advantages over retrospective studies, since they avoid the problems of reverse causality and differences in sample handling, but they are still subject to potential confounding. Studies of the genetic predictors of TL in relation to disease risk avoid these drawbacks, although they are not without limitations. Telomere biology is of major importance to the risk of common disease, but the complexities of the relationship are only now beginning to be understood.This research was supported by Cancer Research UK Programme Awards C588/A10589 and C588/A19167 (MMI and JHB) and C8197/A16565 (AMD and KAP) and the Isaac Newton Trust.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00439-015-1563-

Solvent Effects on Optical Properties of Molecules: A Combined Time-Dependent Density Functional Theory/Effective Fragment Potential Approach

A quantum mechanics/molecular mechanics (QM/MM) type of scheme is employed to calculate the solvent-induced shifts of molecular electronic excitations. The effective fragment potential (EFP) method was used for the classical potential. Since EFP has a density dependent functional form, in contrast with most other MM potentials, time-dependent density functional theory (TDDFT) has been modified to combine TDDFT with EFP. This new method is then used to perform a hybrid QM/MM molecular dynamics simulation to generate a simulated spectrum of the n→π∗ vertical excitation energy of acetone in vacuum and with 100 water molecules. The calculated watersolvent effect on the vertical excitation energy exhibits a blueshift of the n→π∗ vertical excitation energy in acetone (Δω1=0.211 eV), which is in good agreement with the experimental blueshift

Synaptotagmin-Mediated Bending of the Target Membrane Is a Critical Step in Ca2+-Regulated Fusion

SummaryDecades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion