Characterization of tissue transglutaminase 2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in a number of cell functions. Up-regulation of TG2 is thought to be involved in monocyte to macrophage differentiation and defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be fully elucidated. Cell surface-associated TG2 is now recognized as being important in regulating cell adhesion and migration, via its association with cell surface receptors such as syndecan-4, ß1 and ß3 integrin, but its extracellular role in the clearance of apoptotic cells is still not fully explored. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function within the framework of apoptotic cell clearance. Both THP-1 cell-derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors (both cell permeable and impermeable, irreversible and active site directed) resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. Macrophage cell surface TG2 and, in particular, its cell surface crosslinking activity was found to be crucial in dictating apoptotic cell clearance. Our further studies demonstrate syndecan-4 association with TG2 and imply possible cooperation of these proteins in apoptotic cell clearance. Knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance which seems to involve protein cross linking and interaction with other cell surface receptors.

Analysis of Transglutaminase-2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Transglutaminase-2 (TG2) is a Ca2+-dependent protein crosslinking enzyme known to play an important role in apoptotic cell clearance by macrophages through an ill-defined mechanism. Several studies have implicated TG2 in the apoptosis programme e.g. raised TG2 levels in cells undergoing apoptosis; increased cell death with down-regulation of TG2; up-regulation of TG2 in monocytes upon differentiation into macrophages. Defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be elucidated. Here we aim to characterise the role of TG2 in macrophage function with a focus on apoptotic cell clearance. THP-1 monocytes were induced to differentiate to macrophage-like cells by three different stimulants and were analysed for the presence of TG2. Macrophage-apoptotic cell interaction studies in the presence and absence of irreversible TG2 inhibitors resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. TG2 was expressed at the macrophage cell surface and its association with ß3 integrin expression suggests the possible link between TG2 and ß3 integrins. Our current findings suggest that TG2 had got a crucial but yet to be defined role in apoptotic cell clearance.

A study of new product market search and user adoption

The research was carried out in the Aviation Division of Dunlop Limited and was initiated as a search for more diverse uses for carbon/carbon composites. An assumed communication model of adoption was refined by introducing the concept of a two way search after making cross industry comparisons of supplier and consumer behaviour. This research has examined methods of searching for new uses for advanced technology materials. Two broad approaches were adopted. First, a case history approach investigated materials that had been in a similar oosition to carbon/carbon to see how other material producing firms had tackled the problem. Second, a questionnaire survey among industrialists examined: the role and identity of material decision makers in different sized firms; the effectiveness of various information sources and channels; and the material adoption habits of different industries. The effectiveness of selected information channels was further studied by monitoring the response to publicity given to carbon/carbon. A flow chart has been developed from the results of this research which should help any material producing firm that is contemplating the introduction of a new material to the world market. Further benefit to our understanding of the innovation and adoption of new materials would accrue from work in the followino areas: "micro" type case histories; understanding more fully the role of product champions or promoters; investigating the phase difference between incremental and radical type innovations for materials; examining the relationship between the adoption rate of new materials and the advance of technology; studying the development of cost per unit function methods for material selection; and reviewing the benefits that economy of scale studies can have on material developments. These are all suggested areas for further work.

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

Characterization of tissue transglutaminase 2 in macrophage function

Apoptosis is a highly regulated process that removes damaged or unwanted cells in vivo and defective clearance of apoptotic cells by macrophages has significant immunological implications. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. TG2 as a guanosine triphosphate (GTP)-binding or GTP- hydrolyzing protein for mediating signal transduction and as a cell cycle regulator emphasized the importance of this enzyme in aging process. The ubiquitous presence of TG2 compared to the other organ-specific TGases has attracted special attention as a cellular aging device. TG2 activity and expression are known to increase in aging humans suggesting possible involvement in several age-related processes such as decrease in vascular compliance and increased stiffening of conduit arteries, cataract formation, Alzheimer's disease and senescent epidermal keratinocytes. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function. THP-1 cell derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors resulted in significant inhibition of interaction. Macrophage cell surface TG2 and, in particular, its cell surface cross linking activity was found to be crucial in apoptotic cell clearance. Syndecan-4 association with TG2 implies possible cooperation of these proteins and knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance.

Building and applying a concept hierarchy representation of a user profile

Term dependence is a natural consequence of language use. Its successful representation has been a long standing goal for Information Retrieval research. We present a methodology for the construction of a concept hierarchy that takes into account the three basic dimensions of term dependence. We also introduce a document evaluation function that allows the use of the concept hierarchy as a user profile for Information Filtering. Initial experimental results indicate that this is a promising approach for incorporating term dependence in the way documents are filtered.

GEO Label:user and producer perspectives on a label for geospatial data

One of the aims of the Science and Technology Committee (STC) of the Group on Earth Observations (GEO) was to establish a GEO Label- a label to certify geospatial datasets and their quality. As proposed, the GEO Label will be used as a value indicator for geospatial data and datasets accessible through the Global Earth Observation System of Systems (GEOSS). It is suggested that the development of such a label will significantly improve user recognition of the quality of geospatial datasets and that its use will help promote trust in datasets that carry the established GEO Label. Furthermore, the GEO Label is seen as an incentive to data providers. At the moment GEOSS contains a large amount of data and is constantly growing. Taking this into account, a GEO Label could assist in searching by providing users with visual cues of dataset quality and possibly relevance; a GEO Label could effectively stand as a decision support mechanism for dataset selection. Currently our project - GeoViQua, - together with EGIDA and ID-03 is undertaking research to define and evaluate the concept of a GEO Label. The development and evaluation process will be carried out in three phases. In phase I we have conducted an online survey (GEO Label Questionnaire) to identify the initial user and producer views on a GEO Label or its potential role. In phase II we will conduct a further study presenting some GEO Label examples that will be based on Phase I. We will elicit feedback on these examples under controlled conditions. In phase III we will create physical prototypes which will be used in a human subject study. The most successful prototypes will then be put forward as potential GEO Label options. At the moment we are in phase I, where we developed an online questionnaire to collect the initial GEO Label requirements and to identify the role that a GEO Label should serve from the user and producer standpoint. The GEO Label Questionnaire consists of generic questions to identify whether users and producers believe a GEO Label is relevant to geospatial data; whether they want a single "one-for-all" label or separate labels that will serve a particular role; the function that would be most relevant for a GEO Label to carry; and the functionality that users and producers would like to see from common rating and review systems they use. To distribute the questionnaire, relevant user and expert groups were contacted at meetings or by email. At this stage we successfully collected over 80 valid responses from geospatial data users and producers. This communication will provide a comprehensive analysis of the survey results, indicating to what extent the users surveyed in Phase I value a GEO Label, and suggesting in what directions a GEO Label may develop. Potential GEO Label examples based on the results of the survey will be presented for use in Phase II.

Paternal low protein diet affects adult offspring cardiovascular and metabolic function in mice

Although the association between maternal periconceptional diet and adult offspring health is well characterised, our understanding of the impact of paternal nutrition at the time of conception on offspring phenotype remains poorly defined. Therefore, we determined the effect of a paternal preconception low protein diet (LPD on adult offspring cardiovascular and metabolic health in mice. Male C57BL/6 mice were fed either normal protein diet (NPD; 18% casein or LPD (9% casein for 7 wk before mating. At birth, a reduced male-to-female ratio (P = 0.03 and increased male offspring weight (P = 0.009 were observed in litters from LPD compared with NPD stud males with no differences in mean litter size. LPD offspring were heavier than NPD offspring at 2 and 3 wk of age (P <0.02. However, no subsequent differences in body weight were observed. Adult male offspring derived from LPD studs developed relative hypotension (decreased by 9.2 mmHg and elevated heart rate (P <0.05, whereas both male and female offspring displayed vascular dysfunction and impaired glucose tolerance relative to NPD offspring. At cull (24 wk, LPD males had elevated adiposity (P = 0.04, reduced heart-to-body weight ratio (P = 0.04, and elevated circulating TNF-α levels (P = 0.015 compared with NPD males. Transcript expression in offspring heart and liver tissue was reduced for genes involved in calcium signaling (Adcy, Plcb, Prkcb and metabolism (Fto in LPD offspring (P <0.03. These novel data reveal the impact of suboptimal paternal nutrition on adult offspring cardiovascular and metabolic homeostasis, and provide some insight into the underlying regulatory mechanisms.

Polynomial function intervals for floating-point software verification

The focus of our work is the verification of tight functional properties of numerical programs, such as showing that a floating-point implementation of Riemann integration computes a close approximation of the exact integral. Programmers and engineers writing such programs will benefit from verification tools that support an expressive specification language and that are highly automated. Our work provides a new method for verification of numerical software, supporting a substantially more expressive language for specifications than other publicly available automated tools. The additional expressivity in the specification language is provided by two constructs. First, the specification can feature inclusions between interval arithmetic expressions. Second, the integral operator from classical analysis can be used in the specifications, where the integration bounds can be arbitrary expressions over real variables. To support our claim of expressivity, we outline the verification of four example programs, including the integration example mentioned earlier. A key component of our method is an algorithm for proving numerical theorems. This algorithm is based on automatic polynomial approximation of non-linear real and real-interval functions defined by expressions. The PolyPaver tool is our implementation of the algorithm and its source code is publicly available. In this paper we report on experiments using PolyPaver that indicate that the additional expressivity does not come at a performance cost when comparing with other publicly available state-of-the-art provers. We also include a scalability study that explores the limits of PolyPaver in proving tight functional specifications of progressively larger randomly generated programs. © 2014 Springer International Publishing Switzerland.

Monocyte subset counts associations with left ventricular systolic function post ST elevation myocardial infarction

Background: Monocytes are implicated in the initiation and progression of theatherosclerotic plaque contributing to plaque instability and rupture. Little is knownof the role played by the 3 phenotypically and functionally different monocytesubpopulations in determining ventricular remodeling following ST elevation my-ocardial infarction (STEMI). Mon1 are "classical" inflammatory monocytes, whilstMon3 are considered reparative with fibroblast deposition ability. The function ofthe newly described Mon2 is yet to be elucidated. Method: STEMI patients (n=196, mean age 62±13 years; 72% male) treatedwith percutaneous revascularization were recruited within the first 24 hours. Pe-ripheral blood monocyte subpopulations were enumerated and characterizedusing flow cytometry after staining for CD14, CD16 and CCR2. Phenotypi-cally, monocyte subpopulations are defined as: CD14+CD16-CCR2+ (Mon1),CD14+CD16+CCR+ (Mon2) and CD14lowCD16+CCR2- (Mon3) cells. Transtho-racic 2D echocardiography was performed within 7 days and 6 months post infarctto assess ventricular volumes, mass, systolic, and diastolic functions. Results: Using linear regression analysis higher counts for Mon1, and lowercounts for Mon2 and Mon3 were significantly associated with the baseline leftventricular ejection fraction (LVEF) within seven days post infarction. At 6 monthspost STEMI lower counts of Mon2 remained positively associated with decreasedLVEF (p value= 0.002).Monocyte subsets correlation with LVEFMonocytes mean florescence Baseline left ventricular Left ventricular ejectionintensity (cells/μl) ejection fraction (%) fraction (%) at 6 months post infarctβ-value P-valueβ-value P-valueTotal Mon0.31 P<0.001 0.360.009Mon 10.019 0.020.070.62Mon 2−0.28 0.001 −0.420.002Mon 3−0.27 0.001 −0.180.21 Conclusion: Peripheral monocytes of all three subsets correlate with LVEF af-ter a myocardial infarction. High counts of the inflammatory Mon1 are associatedwith reduction in the baseline LVEF. Post remodelling, the convalescent EF wasindependently predicted by monocyte subpopulation 2. As lower counts depictednegative ventricular remodeling, this suggests a reparative role for the newly de-scribed Mon2, possibly via myofibroblast deposition and angiogenesis, in contrastto an anticipated inflammatory role.

Combined quality function deployment and logical framework analysis to improve quality of emergency care in Malta

Purpose – The purpose of this paper is to develop an integrated patient-focused analytical framework to improve quality of care in accident and emergency (A&E) unit of a Maltese hospital. Design/methodology/approach – The study adopts a case study approach. First, a thorough literature review has been undertaken to study the various methods of healthcare quality management. Second, a healthcare quality management framework is developed using combined quality function deployment (QFD) and logical framework approach (LFA). Third, the proposed framework is applied to a Maltese hospital to demonstrate its effectiveness. The proposed framework has six steps, commencing with identifying patients’ requirements and concluding with implementing improvement projects. All the steps have been undertaken with the involvement of the concerned stakeholders in the A&E unit of the hospital. Findings – The major and related problems being faced by the hospital under study were overcrowding at A&E and shortage of beds, respectively. The combined framework ensures better A&E services and patient flow. QFD identifies and analyses the issues and challenges of A&E and LFA helps develop project plans for healthcare quality improvement. The important outcomes of implementing the proposed quality improvement programme are fewer hospital admissions, faster patient flow, expert triage and shorter waiting times at the A&E unit. Increased emergency consultant cover and faster first significant medical encounter were required to start addressing the problems effectively. Overall, the combined QFD and LFA method is effective to address quality of care in A&E unit. Practical/implications – The proposed framework can be easily integrated within any healthcare unit, as well as within entire healthcare systems, due to its flexible and user-friendly approach. It could be part of Six Sigma and other quality initiatives. Originality/value – Although QFD has been extensively deployed in healthcare setup to improve quality of care, very little has been researched on combining QFD and LFA in order to identify issues, prioritise them, derive improvement measures and implement improvement projects. Additionally, there is no research on QFD application in A&E. This paper bridges these gaps. Moreover, very little has been written on the Maltese health care system. Therefore, this study contributes demonstration of quality of emergency care in Malta.

Synthesis and characterisation of interfacial layers in organic solar cells

The quest for renewable energy sources has led to growing attention in the research of organic photovoltaics (OPVs), as a promising alternative to fossil fuels, since these devices have low manufacturing costs and attractive end-user qualities, such as ease of installation and maintenance. Wide application of OPVs is majorly limited by the devices lifetime. With the development of new encapsulation materials, some degradation factors, such as water and oxygen ingress, can almost be excluded, whereas the thermal degradation of the devices remains a major issue. Two aspects have to be addressed to solve the problem of thermal instability: bulk effects in the photoactive layer and interfacial effects at the photoactive layer/charge-transporting layers. In this work, the interface between photoactive layer and electron-transporting zinc oxide (ZnO) in devices with inverted architecture was engineered by introducing polymeric interlayers, based on zinc-binding ligands, such as 3,4-dihydroxybenzene and 8-hydroxyquinoline. Also, a cross-linkable layer of poly(3,4-dimethoxystyrene) and its fullerene derivative were studied. At first, controlled reversible addition-fragmentation chain transfer (RAFT) polymerisation was employed to achieve well-defined polymers in a range of molar masses, all bearing a chain-end functionality for further modifications. Resulting polymers have been fully characterised, including their thermal and optical properties, and introduced as interlayers to study their effect on the initial device performance and thermal stability. Poly(3,4-dihydroxystyrene) and its fullerene derivative were found unsuitable for application in devices as they increased the work function of ZnO and created a barrier for electron extraction. On the other hand, their parental polymer, poly(3,4-dimethoxystyrene), and its fullerene derivative, upon cross-linking, resulted in enhanced efficiency and stability of devices, if compared to control. Polymers based on 8-hydroxyquinoline ligand had a negative effect on the initial stability of the devices, but increased the lifetime of the cells under accelerated thermal stress. Comprehensive studies of the key mechanisms, determining efficiency, such as charge generation and extraction, were performed by using time-resolved electrical and spectroscopic techniques, in order to understand in detail the effect of the interlayers on the device performance. Obtained results allow deeper insight into mechanisms of degradation that limit the lifetime of devices and prompt the design of better materials for the interface stabilisation.

The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.