Bone marrow derived cells as endothelial precursors and the role of multi-potent progenitor cells in repairing ischaemic tissues.

PhDIntroduction: Atherosclerosis and its complications are a major cause of death and disability and it remains a major challenge to develop new therapies for patients with irreversible end organ damage and ongoing ischaemia. The discovery of adult stem and progenitor cells with the ability to regenerate adult tissues holds great promise. Bone marrow is the source of both endothelial progenitor cells (EPCs) and multi-potent adult progenitor cells (MAPCs). MAPCs are rare pluripotent bone marrow derived cells with the theoretical potential to differentiate into tissues of all three germ cell layers, including endothelium. These cells may have the potential to facilitate cardiac repair. The aim of this thesis was to further characterise bone marrow derived endothelial progenitor cells including multi potent adult progenitor cells and assess their angiogenic potential and mechanisms of action in animal models of cardiovascular disease. Findings: EPCs were isolated from humans and mice and their phenotype, markers and function determined, including gene tracking experiments in mice utilising the Cre/Lox system. It was not possible to isolate cells with the same phenotype as MAPCs from rodent bone marrow. However, cells with pluri-potent properties, named rat multi-potent progenitor cells (rMPCs), were isolated from rat bone marrow. These cells had the ability to up regulate tissue specific antigens from all 3 germ cell lineages and in addition secreted multiple cytokines related to angiogenesis and inflammation. To investigate the in vivo properties of rMPCs a rat hind limb model of ischaemia was established and syngeneic rMPCs were transplanted into the ischaemic hind limbs. rMPCs engrafted selectively into the adventitia of arterioles of ischaemic muscles. However, engrafted cells did not differentiate into an endothelial or smooth muscle phenotype. Cytokine analysis of muscles 5 days after rMPC injection revealed raised levels of cytokines, including chemokines MCP1 and SDR. Limb perfusion, measured by microspheres, increased after rMPC injection. In addition a novel MRI based assessment of ischaemic muscles revealed a significant normalisation of MRI signal after rMPC transplantation. However, there was no improvement in limb function assessed by treadmill running distance 4 weeks after cell injection. These findings suggest that transplantation of rMPCs into ischaemic muscles may modulate local inflammatory and angiogenic responses through paracrine mechanisms. Conclusion: Despite the potential for stem and progenitor cells to be used for the treatment of chronic cardiac ischaemia the biology of stem cells is still relatively poorly understood, as is the mechanism of action of cells after transplantation. As set out in the aims, the work in this thesis adds further to our understanding of both EPCs and BM derived pluri-potent stem cells. In addition it provides insight into the hind limb ischaemia model and the mechanism of action of cell therapy after transplantation into ischaemic muscle

Clinical value of chest pain presentation and prodromes on the assessment of cardiovascular disease: a cohort study.

OBJECTIVES: The recognition of coronary artery disease (CAD) among patients who report chest pain remains difficult in primary care. This study investigates the association between chest pain (specified, unspecified or musculoskeletal) and prodromes (dyspepsia, fatigue or dyspnoea), with first-ever acute CAD, and increased longer term cardiovascular risk. DESIGN: Cohort study. SETTING: Anonymised clinical data recorded electronically by general practitioners from 140 primary care surgeries in London (UK) between April 2008 and April 2013. PARTICIPANTS: Data were extracted for all patients aged 30 years and over at the beginning of the study period, registered in the surgeries. MAIN OUTCOME MEASURES: Clinical data included chest pain, dyspepsia, dyspnoea and fatigue, first-ever CAD and long-term cardiovascular risk (QRisk2). Regression models were used to analyse the association between chest pain together with prodromes and CAD and QRisk2≥20%. RESULTS: 354,052 patients were included in the study. 4842 patients had first-ever CAD of which 270 reported chest pain in the year before the acute event. 257,019 patients had QRisk2 estimations. Chest pain was associated with a higher risk of CAD. HRs: 21.12 (16.68 to 26.76), p<0.001; 7.51 (6.49 to 8.68), p<0.001; and 1.84 (1.14 to 3.00), p<0.001 for specified, unspecified and musculoskeletal chest pain. Dyspepsia, dyspnoea or fatigue was also associated with a higher risk of CAD. Chest pain of all subtypes, dyspepsia and dyspnoea were also associated with an increased 10-year cardiovascular risk of 20% or more. CONCLUSIONS: All patients with chest pain, including those with atypical symptoms, require careful assessment for acute and longer term cardiovascular risk. Prodromes may have independent diagnostic value in the estimation of cardiovascular disease risk

Determination of the nature of the Cu coordination complexes formed in the presence of NO and NH3 within SSZ-13

Ammonia-selective catalytic reduction (NH3-SCR) using Cu zeolites is a well-established strategy for the abatement of NOx gases. Recent studies have demonstrated that Cu is particularly active when exchanged into the SSZ-13 zeolite, and its location in either the 6r or 8r renders it an excellent model system for fundamental studies. In this work, we examine the interaction of NH3-SCR relevant gases (NO and NH3) with the Cu2+ centers within the SSZ-13 structure, coupling powder diffraction (PD), X-ray absorption spectroscopy (XAFS), and density functional theory (DFT). This combined approach revealed that, upon calcination, cooling and gas exposure Cu ions tend to locate in the 8r window. After NO introduction, Cu-ions are seen to coordinate to two framework oxygens and one NO molecule, resulting in a bent Cu-nitrosyl complex with a Cu-N-O bond angle of similar to 150 degrees. Whilst Cu seems to be partially reduced/changed in coordination state, NO is partially oxidized. On exposure to NH3 while the PD data suggest the Cu2+ ion occupies a similar position, simulation and XAFS pointed toward the formation of a Jahn-Teller distorted hexaamine complex [Cu(NH3)(6)](2+) in the center of the cha cage. These results have important implications in terms of uptake and storage of these reactive gases and potentially for the mechanisms involved in the NH3-SCR process

Intra-promoter switch of transcription initiation sites in proliferation signaling-dependent RNA metabolism

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.</p

The discovery, abundances and [Fe/H]NB392 distributions of metal-poor stars in the halo of the Milky Way

By boldly looking where no one has looked before, this study surveyed a volume within the Milky Way halo to discover population III stars or their direct descendants. A cat- alogue of 123,505 sources was obtained by surveying the COSMOS field with the WFC and the NB392 filter (λc=3918 ̊A, ∆λ=52 ̊A), sensitive to CaHK. A star-galaxy cut was devised from samples of confirmed stars and galaxies based on spectroscopic redshifts and the apparent optical and infra-red colours of the samples, with a completeness and contamination of 75.3% and 6.1% respectively. The catalogue was reduced to 338 potential stars by introducing a limiting magnitude of 24.7, making it 5-7 magni- tudes deeper than the Pristine survey and corresponding to a limiting distance of 166 kpc for G-type stars. This survey covered 1.89 deg2, corresponding to volumes from (1.89 × 108)pc3 to (8.55 × 1016)pc3 from spectral types M to O. Stars with known metallicity, [Fe/H], values from the POLLUX database were used to create an [Fe/H] (NB392−u) − (g − i) by (g − i) grid and the 338 candidates were assigned metallicity values. This created a final catalogue that was sorted by metallicity and candidates with [Fe/H]<−3 were visually inspected using the HST F814W filter to determine whether they were stars, in which seven were found. These could give an insight to the earliest stars in the universe. Number densities were calculated for F, G and K-type stars with [Fe/H] values of −4 to be (4.3±3.0)×10−14 pc −3, (1.36±0.96)×10−13 pc −3 and (3.0 ± 2.1) × 10−11 pc −3 respectively. It was found that the number density of stars with [Fe/H]<−3 is less than expected by a factor of ∼ 10.8, however it is ≈ 6.3 times larger for stars with [Fe/H]<−4

RMT: R-matrix with time-dependence. Solving the semi-relativistic, time-dependent Schrödinger equation for general, multi-electron atoms and molecules in intense, ultrashort, arbitrarily polarized laser pulses

RMT is a program which solves the time-dependent Schrödinger equation for general, multielectron atoms, ions and molecules interacting with laser light. As such it can be used to model ionization (single-photon, multiphoton and strong-field), recollision (high-harmonic generation, strong-field rescattering) and, more generally, absorption or scattering processes with a full account of the multielectron correlation effects in a time-dependent manner. Calculations can be performed for targets interacting with ultrashort, intense laser pulses of long wavelength and arbitrary polarization. Calculations for atoms can optionally include the Breit–Pauli correction terms for the description of relativistic (in particular, spin–orbit) effects

Development of a 3D tissue-engineered skeletal muscle and bone co‐culture system

In vitro three‐dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone were investigated. High‐glucose Dulbecco's Modified Eagle Medium (HG‐DMEM) supplemented with 20% foetal bovine serum (FBS) followed by HG‐DMEM with 2% horse serum was found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an up‐regulation of RUNX2/CBFa1 in TE85s. Myotube formation was also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs had significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appeared reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step towards a full in vitro 3D musculoskeletal junction model

Validation of the CREST score for predicting circulatory-aetiology death in out-of-hospital cardiac arrest without STEMI

Aims: The CREST tool was recently developed to stratify the risk of circulatory-aetiology death (CED) in out-of-hospital cardiac arrest (OHCA) patients without ST-elevation myocardial infarction (STEMI). We aimed to validate the CREST score using an external cohort and determine whether it could be improved by the addition of serum lactate on admission. Methods: The study involved the retrospective analysis of consecutive patients admitted to a single tertiary centre with OHCA of presumed cardiac origin over a 51-month period. The CREST score was calculated by attributing points to the following variables: Coronary artery disease (CAD), non-shockable Rhythm, Ejection fraction <30%, cardiogenic Shock at presentation and ischaemic Time ≥25 minutes. The primary endpoint was CED vs neurological aetiology death (NED) or survival. Results: Of 500 patients admitted with OHCA, 211 did not meet criteria for STEMI and were included. 115 patients died in hospital (71 NED, 44 CED). When analysed individually, CED was associated with all CREST variables other than a previous diagnosis of CAD. The CREST score accurately predicted CED with excellent discrimination (C-statistic 0.880, 95% CI 0.813-0.946) and calibration (Hosmer and Lemeshow P=0.948). Although an admission lactate ≥7 mmol/L also predicted CED, its addition to the CREST score (the C-AREST score) did not significantly improve the predictive ability (CS 0.885, 0.815-0.954, HS P=0.942, X2 difference in -2 log likelihood =0.326, P=0.850). Conclusion: Our study is the first to independently validate the CREST score for predicting CED in patients presenting with OHCA without STEMI. Addition of lactate on admission did not improve its predictive ability.Publisher PDFPeer reviewe