The application of laser-generated ultrasound to the study of aluminium-epoxy bonded systems

The poor performance of acoustic wave techniques in predicting adhesively-bonded joint failure under destructive loading is a long-standing problem, known to derive from unreliable adhesive defect detection. This thesis examines the feasibility of applying a relatively new technique, generating ultrasound with pulsed Nd:YAG lasers, to the study of aluminium alloy adherends joined by epoxy layer bonds. Laser generation is a non-contacting method which produces highly repeatable ultrasonic sources in metals, without damping motion at the sample surface. Pulses created in this fashion have bandwidths around 20 MHz and radiate both along the sample surface and into the material bulk. Displacements at the sample surface recorded by a broad bandwidth non-contact detector, such as a 532 nm wavelength laser Michelson interferometer, are therefore able to resolve details in time-varying traces which are not visible when narrowband transducers are used. In particular, individual reverberations between the interfaces of epoxy layers less than 100 μm thick are detected in transmission through adhesively bonded joints, on time domain traces. An epoxy layer sandwiched between two thick aluminium adherends presents a three layer case which is seldom discussed in the literature. I have therefore adapted theory developed for surface waves in thin layers overlying deep substrates, and for waves transmitted through multilayer structures, into an explicit formulation for an elastic layer embedded in adherend half-spaces that can be used for both through-transmission and interface-parallel waves. The case of travelling waves in a viscoelastic layer has not yet been examined as the current formulation requires unfeasibly long computation times. A numerical solution assuming elastic behaviour, gives strong indications that embedded epoxy layers support travelling waves directed along the interfaces, despite the fact that a single interface between epoxy and aluminium will not support non-dispersive Stoneley interface waves. Experimental work presented in Chapters 4 to 7 is preceded by a review of laser generation and non-contact detection methods, which introduces techniques that I have employed. As well as using laser interferometers, 1 have also built my own electromagnetic acoustic transducers (EMATs), to provide a cheaper alternative detection scheme. Chapter 4 concentrates upon on-epicentre detection of direct through transmission pulse arrivals, using analysis both of the entire reverberation wavetrain following the main arrival and of consecutive pulses within it, in order to extract information on the bonds' cohesive and adhesive properties. Chapter Five examines variations in surface-travelling waveforms on unbonded, free-surface aluminium plates with thicknesses varying from 63 mm down 28 μm, in a search for non- dispersive waves that would be suitable for probing adhesive bonds. Rayleigh arrivals on samples over 10 mm thick and the symmetric zero-order Lamb mode on plates under 200 μm thick both propagate from the NdtYAG laser source as sharp pulses, but intermediate plate thicknesses only allow waves with highly dispersive characteristics, which tend to mask any dispersion due to bonds. The plate wave experiments allow a full intercomparison between interferometer and EMATs, both out-of-plane motion sensitive and in-plane motion sensitive. Chapter 6 uses Rayleigh-like surface waves travelling along 25 mm thick adherends to initiate interface-parallel travelling waves in an adhesive layer bonding a second adherend to the surface, which are subsequently detected on emerging at the free surface beyond the bond. These surface-interface-surface travelling (SIST) waves penetrate under increasingly longer bonds as the wave frequency decreases, a fact confirmed by the behaviour of pulses given a narrowband frequency modulation when generating laser beams interfere to produce a spatially modulated source. The interference source optical arrangement, described in Chapter 5, can be altered to give Rayleigh arrival modulation frequencies from 20 MHz to below 1 MHz. Finally, Chapter 7 examines alternative pathways for surface waves incident upon the edge of a bonded joint region, and demonstrates that SIST waves are an efficient mechanism for transferring ultrasound between the two adhesive-adherend interfaces, given the observed emergence of clearly discernible SIST waves on the second adherend of a lapped bond joint. I conclude that through transmission pulse analyses arc capable of extracting quantitative information about bond properties and should be developed as the basis for laser generated ultrasonic bond testing. SIST waves, however, require further research before they can be employed in a practical manner

RNA helicase EIF4A1-mediated translation is essential for the GC response

EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5' untranslated regions. However, very little is known of their roles in nonmalignant cells. Using mouse models to delete Eif4a1, Eif4b or Eif4h in B cells, we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. After B cell activation in vitro, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression, and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable, whereas inhibition of EIF4A1 and EIF4A2 by Hippuristanol treatment induces cell death.</p

RNA helicase EIF4A1-mediated translation is essential for the GC response

EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5' untranslated regions. However, very little is known of their roles in nonmalignant cells. Using mouse models to delete Eif4a1, Eif4b or Eif4h in B cells, we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. After B cell activation in vitro, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression, and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable, whereas inhibition of EIF4A1 and EIF4A2 by Hippuristanol treatment induces cell death.</p

The human insulin receptor mRNA contains a functional internal ribosome entry segment.

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective

Single cell tracking of gadolinium labeled CD4(+) T cells by laser ablation inductively coupled plasma mass spectrometry

Cellular therapy is emerging as a promising alternative to conventional immunosuppression in the fields of haematopoietic stem cell (HSC) transplantation, autoimmune disease and solid organ transplantation. Determining the persistence of cell-based therapies in vivo is crucial to understanding their regulatory function and requires the combination of an extremely sensitive detection technique and a stable, long-lifetime cell labelling agent. This paper reports the first application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform single cell detection of T cell populations relevant to cellular immunotherapy. Purified human CD4+ T cells were labelled with commercially available Gd-based MRI contrast agents, Omniscan® and Dotarem®, which enabled passive loading of up to 108 Gd atoms per cell. In mixed preparations of labelled and unlabelled cells, LA-ICP-MS was capable of enumerating labelled cells at close to the predicted ratio. More importantly, LA-ICP-MS single cell analysis demonstrated that the cells retained sufficient label to remain detectable for up to 10 days post-labelling both in vitro and in vivo in an immunodeficient mouse model

Absent expansion of AXIN2+ hepatocytes and altered physiology in Axin2CreERT2 mice challenges the role of pericentral hepatocytes in homeostatic liver regeneration

Background &amp; Aims: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. Methods: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. Results: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/β-catenin signalling and related metabolomic disturbance. Conclusions: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. Impact and implications: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally

Teleconnections of the Quasi‐Biennial Oscillation in a multi‐model ensemble of QBO‐resolving models

The Quasi-biennial Oscillation (QBO) dominates the interannual variability of the tropical stratosphere and influences other regions of the atmosphere. The high predictability of the QBO implies that its teleconnections could lead to increased skill of seasonal and decadal forecasts provided the relevant mechanisms are accurately represented in models. Here modelling and sampling uncertainties of QBO teleconnections are examined using a multi-model ensemble of QBO-resolving atmospheric general circulation models that have carried out a set of coordinated experiments as part of the Stratosphere-troposphere Processes And their Role in Climate (SPARC) QBO initiative (QBOi). During Northern Hemisphere winter, the stratospheric polar vortex in most of these models strengthens when the QBO near 50 hPa is westerly and weakens when it is easterly, consistent with, but weaker than, the observed response. These weak responses are likely due to model errors, such as systematically weak QBO amplitudes near 50 hPa, affecting the teleconnection. The teleconnection to the North Atlantic Oscillation is less well captured overall, but of similar strength to the observed signal in the few models that do show it. The models do not show clear evidence of a QBO teleconnection to the Northern Hemisphere Pacific-sector subtropical jet

The Met Office Unified Model Global Atmosphere 6.0/6.1 and JULES Global Land 6.0/6.1 configurations

We describe Global Atmosphere 6.0 and Global Land 6.0: the latest science configurations of the Met Office Unified Model and JULES land surface model developed for use across all timescales. Global Atmosphere 6.0 includes the ENDGame dynamical core, which significantly increases mid-latitude variability improving a known model bias. Alongside developments of the model’s physical parametrisations, ENDGame also increases variability in the tropics, which leads to an improved representation of tropical cyclones and other tropical phenomena. Further developments of the atmospheric and land surface parametrisations improve other aspects of model performance, including the forecasting of surface weather phenomena. We also describe Global Atmosphere 6.1 and Global Land 6.1, which include a small number of long-standing differences from our main trunk configurations that we continue to require for operational global weather prediction. Since July 2014, GA6.1/GL6.1 has been used by the Met Office for operational global NWP, whilst GA6.0/GL6.0 was implemented in its remaining global prediction systems over the following year

Genome-Wide Discovery of Somatic Regulatory Variants in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.&nbsp