Nanoparticle theranostics targeting CLEC14A on tumour vasculature

This thesis studies the development of a method to knockdown CLEC14A in-vivo. CLEC14A has been shown to have strong pro-angiogenic signalling properties and its knockdown in-vitro has been shown to decrease wound healing and tube formation. Additionally, CLEC14A knockout mice showed a decreased rate of tumour growth in implanted Lewis Lung Carcinoma tumours. siRNA is a common tool used to knockdown genes but needs protection from degrading enzymes found in serum. To this end, a chitosan-based nanoparticle was developed to deliver siRNA. A 60% knockdown of CLEC14A was achieved in-vitro using untargeted chitosan nanoparticles. Microarray analysis of HUVEC cells treated with siRNA entrapped nanoparticles showed that CLEC14A was not knocked down at an mRNA level but that endothelial genes related to blood flow were affected in a similar manner to an increase in laminar blood flow, suggesting that CLEC14A has a regulatory role in endothelial gene expression. Conjugating antibodies to the surface of the chitosan nanoparticles may increase cellular uptake and improve knockdown of CLEC14A. Antibody targeting of the nanoparticles did not improve knockdown, and in fact, decreased efficiency at higher concentrations. Bio-distribution studies were performed with untargeted nanoparticles and showed localisation to tumour vascular endothelium. Nanoparticles were found in the liver and kidneys as well as the tumour. An anti-angiogenic, CLEC14A based, vaccine was developed to be delivered by the chitosan nanoparticles. A CLEC14A-Tetanus FrC fusion protein was produced in HEK293 cells but failed to fold correctly and be excreted at a sufficient concentration. A CLEC14A-VLRB fusion was created and cloned into a bacterial plasmid. Two fragments of CLEC14A were created to fuse to VLRB with the small fragment being successfully synthesised and purified ready for in-vivo immunisation studies. Research data from Appendix 5 "Chitosan nanoparticle (CLEC14A siRNA) knockdown in HUVEC: microarray analysis" can be found on the University of Birmingham eData repository at: https://doi.org/10.25500/edata.bham.0000041

Preliminary testing: The devil of statistics?

In quantitative research, the selection of the most appropriate statistical test for the comparison of two-independent samples can be problematic. There is a lack of consensus in the Statistics community regarding the appropriate approach; particularly towards assessing assumptions of normality and equal variances. The lack of clarity in the appropriate strategy affects the reproducibility of results. Statistical packages performing different tests under the same name, only adds to this issue.The process of preliminary testing assumptions of a test using the sample data, before performing a test conditional upon the preliminary test, is performed by some researchers; this practice is often criticised in the literature. Preliminary testing is typically performed at the arbitrary 5% significance level. In this paper this process is reviewed, and additional results are given using simulation, examining a procedure with normality and equal variance preliminary tests

Evaluation of Soilless Media Used in Tobacco Float Systems

A wide range of soilless media is currently available to tobacco growers utilizing the float system for transplant production. Most of the media are predominantly made up of peat moss with varying amounts of perlite, vermiculite,and coconut fibers (coir). One of the most difficult problems for growers has been inconsistency in the media from year to year. Because peat is a natural product, some year to year variability is unavoidable. However, many manufacturers of soilless media have procedures in place to ensure that the final product is as consistent as possible

“Burn Down” Management of Winter Cereal Cover Crops for No-tillage Burley Tobacco Production

Recent developments in the design of no-till transplanters and significant improvements in weed control have made no-till tobacco production a feasible option for burley tobacco growers. No-till production reduces soil erosion when tobacco is grown on sloping land. This helps maintain the long term productivity of the soil and may provide the grower with more options for crop rotation, by allowing sloping land to be utilized for tobacco production

Magnetic Field Evolution in Merging Clusters of Galaxies

We present initial results from the first 3-dimensional numerical magnetohydrodynamical (MHD) simulations of magnetic field evolution in merging clusters of galaxies. Within the framework of idealized initial conditions similar to our previous work, we look at the gasdynamics and the magnetic field evolution during a major merger event in order to examine the suggestion that shocks and turbulence generated during a cluster/subcluster merger can produce magnetic field amplification and relativistic particle acceleration and, as such, may play a role in the formation and evolution of cluster-wide radio halos. The ICM, as represented by the equations of ideal MHD, is evolved self-consistently within a changing gravitational potential defined largely by the collisionless dark matter component represented by an N-body particle distribution. The MHD equations are solved by the Eulerian, finite-difference code, ZEUS. The particles are evolved by a standard particle-mesh (PM) code. We find significant evolution of the magnetic field structure and strength during two distinct epochs of the merger evolution.Comment: 21 pages, 7 figures, Figure 2 is color postscript. Accepted for publication in Ap