The RGBarrier assay: the parallel study of gene regulatory element performance at defined chromosomal locations

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

Heat transfer performance investigation of nanofluids flow in pipe

Different types of base fluids, such as water, engine oil, kerosene, ethanol, methanol, ethylene glycol etc. are usually used to increase the heat transfer performance in many engineering applications. But these conventional heat transfer fluids have often several limitations. One of those major limitations is that the thermal conductivity of each of these base fluids is very low and this results a lower heat transfer rate in thermal engineering systems. Such limitation also affects the performance of different equipments used in different heat transfer process industries. To overcome such an important drawback, researchers over the years have considered a new generation heat transfer fluid, simply known as nanofluid with higher thermal conductivity. This new generation heat transfer fluid is a mixture of nanometre-size particles and different base fluids. Different researchers suggest that adding spherical or cylindrical shape of uniform/non-uniform nanoparticles into a base fluid can remarkably increase the thermal conductivity of nanofluid. Such augmentation of thermal conductivity could play a more significant role in enhancing the heat transfer rate than that of the base fluid. Nanoparticles diameters used in nanofluid are usually considered to be less than or equal to 100 nm and the nanoparticles concentration usually varies from 5% to 10%. Different researchers mentioned that the smaller nanoparticles concentration with size diameter of 100 nm could enhance the heat transfer rate more significantly compared to that of base fluids. But it is not obvious what effect it will have on the heat transfer performance when nanofluids contain small size nanoparticles of less than 100 nm with different concentrations. Besides, the effect of static and moving nanoparticles on the heat transfer of nanofluid is not known too. The idea of moving nanoparticles brings the effect of Brownian motion of nanoparticles on the heat transfer. The aim of this work is, therefore, to investigate the heat transfer performance of nanofluid using a combination of smaller size of nanoparticles with different concentrations considering the Brownian motion of nanoparticles. A horizontal pipe has been considered as a physical system within which the above mentioned nanofluid performances are investigated under transition to turbulent flow conditions. Three different types of numerical models, such as single phase model, Eulerian-Eulerian multi-phase mixture model and Eulerian-Lagrangian discrete phase model have been used while investigating the performance of nanofluids. The most commonly used model is single phase model which is based on the assumption that nanofluids behave like a conventional fluid. The other two models are used when the interaction between solid and fluid particles is considered. However, two different phases, such as fluid and solid phases is also considered in the Eulerian-Eulerian multi-phase mixture model. Thus, these phases create a fluid-solid mixture. But, two phases in the Eulerian-Lagrangian discrete phase model are independent. One of them is a solid phase and the other one is a fluid phase. In addition, RANS (Reynolds Average Navier Stokes) based Standard κ-ω and SST κ-ω transitional models have been used for the simulation of transitional flow. While the RANS based Standard κ-ϵ, Realizable κ-ϵ and RNG κ-ϵ turbulent models are used for the simulation of turbulent flow. Hydrodynamic as well as temperature behaviour of transition to turbulent flows of nanofluids through the horizontal pipe is studied under a uniform heat flux boundary condition applied to the wall with temperature dependent thermo-physical properties for both water and nanofluids. Numerical results characterising the performances of velocity and temperature fields are presented in terms of velocity and temperature contours, turbulent kinetic energy contours, surface temperature, local and average Nusselt numbers, Darcy friction factor, thermal performance factor and total entropy generation. New correlations are also proposed for the calculation of average Nusselt number for both the single and multi-phase models. Result reveals that the combination of small size of nanoparticles and higher nanoparticles concentrations with the Brownian motion of nanoparticles shows higher heat transfer enhancement and thermal performance factor than those of water. Literature suggests that the use of nanofluids flow in an inclined pipe at transition to turbulent regimes has been ignored despite its significance in real-life applications. Therefore, a particular investigation has been carried out in this thesis with a view to understand the heat transfer behaviour and performance of an inclined pipe under transition flow condition. It is found that the heat transfer rate decreases with the increase of a pipe inclination angle. Also, a higher heat transfer rate is found for a horizontal pipe under forced convection than that of an inclined pipe under mixed convection.