Mutational Analysis of the Export Targeting Motif of Fibroblast Growth Factor 2, a Mediator of Tumor-Induced Angiogenesis

The majority of secretory proteins is exported from mammalian cells by the classical secretory pathway involving subcellular compartments such as the endoplasmic reticulum (ER) and the Golgi apparatus. However, basic fibroblast growth factor (FGF2), a potent mediator of tumor-induced angiogenesis, has been shown to be secreted by a non-classical pathway that does not depend on the functions of the ER and the Golgi apparatus. The molecular characterization of the FGF2 export mechanism is not only a fundamental problem in cell biology but also of great interest for biomedical research since it may pave the way for the development of a novel class of anti-angiogenic drugs. In this thesis, a robust model system designed to quantitatively assess FGF2 secretion under various experimental conditions was developed. A retroviral expression system was established in CHO cells that allows for a stable integration of reporter constructs whose expression can be induced by doxicycline. In order to monitor expression of FGF2 reporter molecules they were constructed as GFP fusion proteins. Based on this experimental system, secretion of FGF2-GFP can be quantified by flow cytometry, confocal microscopy and biochemical methods since exported FGF2-GFP binds to cell surface heparan sulfate proteoglycans and, therefore, is accessible by membrane-impermeable tools such as antibodies and biotinylation reagents. In the second part of this thesis, a systematic mutational analysis of the FGF2 open reading frame was conducted in order to identify cis elements that direct FGF2 to its export machinery. Initial experiments revealed the identification of FGF2 mutants that are defective in binding to heparan sulfate proteoglycans. Such mutants were neither detectable on the cell surface nor in the medium of cells suggesting that the interaction of FGF2 with heparan sulfate proteoglycans does not only play a role in FGF2 signaling but also in the overall process of FGF2 externalization from mammalian cells. A collection of more than a hundred FGF2 mutants and corresponding stable cell lines described in this thesis now provide a basis for future studies in order to conduct a detailed analysis of determinants required for FGF2 secretion

Detecting GSR Indicative Particles on Decayed Bones using a Novel Field Kit

Decomposed human remains are complex forensic puzzles, escalating in difficulty as the remains’ age obscures evidence, like trauma. Research has shown that scanning electron microscopes with energy dispersive X-ray spectrometers (SEM-EDX) are capable of detecting and identifying gunshot residue (GSR) particles on bones. However, SEM-EDX work is time consuming, expensive, and not accessible to every forensic department. Therefore, a preliminary field test capable of detecting GSR indicative particles, like lead, could save departments money and assist in trauma identification. This study examines the viability of using either the 3M Lead Check Test swabs or a sodium rhodizonate solution as part of a field test to detect lead, a GSR indicative particle, on shot pork ribs that have been allowed to decay. Of the 60 buried ribs, 50 were recovered, and 25 ribs each were tested with the 3M Test Swab or the sodium rhodizonate solution. The success rate of the 3M swabs was much higher when compared to the sodium rhodizonate solution; 64% of samples tested positive for lead using the 3M swabs versus the 3.8% of samples that tested positive for lead with the sodium rhodizonate solution. The 3M swabs allowed for a more direct application to the trauma site and interreacted better with the bone medium than the sodium rhodizonate. The experiment shows that a preliminary field kit test shows promise in helping identify trauma that could be either ballistic or blunt force in nature, but further refinement of the test is needed before recommending it for use

Assessing the regional impact of Indonesian biomass burning emissions based on organic molecular tracers and chemical mass balance modeling

Biomass burning activities commonly occur in Southeast Asia (SEA), and are particularly intense in Indonesia during the dry seasons. The effect of biomass smoke emissions on air quality in the city state of Singapore was investigated during a haze episode in October 2006. Substantially increased levels of airborne particulate matter (PM) and associated chemical species were observed during the haze period. Specifically, the enhancement in the concentration of molecular tracers for biomass combustion such as levoglucosan by as much as two orders of magnitude and the diagnostic ratios of individual organic compounds indicated that biomass burning emissions caused a regional smoke haze episode due to their long-range transport by prevailing winds. With the aid of air mass backward trajectories and chemical mass balance modeling, large-scale forest and peat fires in Sumatra and Kalimantan were identified as the sources of the smoke aerosol, exerting a significant impact on air quality in downwind areas, such as Singapore

Hazardous aerosol emissions during agriculture biomass burning season in Son La and Ba Vi regions, Vietnam

Major national emission sources are assessed by characterization of smoke pollution arising due to traditional agriculture, domestic, and cooking activities in the regions of the biggest biomass burning. Measurement campaigns were carried in Son La and Ba Vi regions, Vietnam, during the dry seasons of 2013 and 2015-2016. PM and BC monitoring, aerosol sampling, chemical speciation were conducted to evaluate ambient smoke level, to relate the characteristics of local on-field emissions to regional aerosols, and to identify the dangerous components of smoke composition. The regions Son La and Ba Vi in February-June faced severe levels of air pollution, with critical PM2.5 and PM10 concentrations up to 130 and 167 µg/m3, respectively, significantly exceeding the air quality standards. A wide range of PM mass concentrations was categorized according to the smoke level, supported by the evolution of carbon (OC, EC) fractions as well as ionic species and molecular markers. The level of PM and BC concentrations was seen to be dependent on factors such as weather conditions and precipitation. Non-acid carbonyls, carboxylates, and aliphatic carbon compounds were evolved with increasing smoke intensity, together with carbonates in coarse size fractions, indicating a large impact of smoke emissions and soil lifted up by the intense fires. On-field emissions in both smoldering and flaming phases were assessed in near-source measurements

Dying in hospital in Ireland: an assessment of the quality of care in the last week of life: National audit of end-of-life care in hospitals in Ireland, 2008/9

The context of this report is set by the fact that most people die in a hospital or similar setting, outside the home. When you consider that most people are also born in hospital, and may spend some time there over the course of a lifetime, it becomes clear that hospitals are central to our passage into life and out of it, touching people at the most important and intimate moments of their lives. In this sense, the work of hospitals mirrors the cycle of life and the expectations of society about its role at each stage of the life cycle. The report assesses the quality of care provided by Irish hospitals in the last week of life. The word ‘hospital’ shares a common linguistic root with words like hospice and hospitality. Hospitality – understood as being welcomed and cared for with kindness and attentiveness - is still what everyone seeks when they come to hospital, including patients and their families who are going through the journey of dying, death and bereavement. That is why the Hospice Friendly Hospitals Programme (2007-2012) commissioned this first ever national audit of endof-life care in Irish hospitals. This report contributes to the growing practice within the Irish hospital system of auditing performance against standards in order to ensure that every aspect of its work meets, and even exceeds, the highest standards of care and excellence. Given that end-of-life care standards did not exist at the time the audit – but have since been published as Quality Standards for End-of-Life Care in Hospitals1 – it may be more appropriate to regard this report as a ‘pre-audit’ or ‘baseline-audit’. It is Government policy, since February 2009, to introduce a mandatory licensing system whereby each hospital will only be allowed to practice if, on the basis of audited performance, it meets acceptable quality standards of service

Implications of regional surface ozone increases on visibility degradation in southeast China

Long-term visibility (1968–2010) and air pollutant (1984–2010) data records in Hong Kong reveal that the occurrence of reduced visibility (RV, defined as the percentage of hours per month with visibility below 8 km in the absence of rain, fog, mist or relative humidity above 95%) in southeast China has increased significantly in the last four decades. The most pronounced rate of increase was observed after 1990 (nine times higher than that before 1990), when notable increases in surface ozone (O3) levels were simultaneously observed (1.06 µg m−3 per yr). The greatest increases in RV, and in O3, NO2 and SO2 concentrations are coincident in the autumn (1.47, 0.20 and 0.45 µg m−3 per yr respectively), when southeast China is strongly influenced by regional O3 formation and accumulation due to continental outflow of pollution from the east China coast under favourable meteorological conditions. Multiple regression revealed that the RV percentage correlated well (p<0.05) with NO2 and NO x in the 1980s, and with NO2, SO2 and O3 after the 1990s, suggesting that there have been changes in the predominant factors causing visibility degradation. In order to elucidate the reasons for these changes, the results were integrated with data from previous research. Possible impacts of elevated O3 on secondary particle formation and their effects on visibility degradation and aerosol radiative forcing in an oxidant-enhanced southeast China are highlighted. Other factors potentially leading to visibility degradation, such as ship emissions and biomass burning, are also discussed

Dying in Hospital in Ireland: An Assessment of the Quality of Care in the Last Week of Life, Final Synthesis Report

The context of this report is set by the fact that most people die in a hospital or similar setting, outside the home. When you consider that most people are also born in hospital, and may spend some time there over the course of a lifetime, it becomes clear that hospitals are central to our passage into life and out of it, touching people at the most important and intimate moments of their lives. In this sense, the work of hospitals mirrors the cycle of life and the expectations of society about its role at each stage of the life cycle.The report assesses the quality of care provided by Irish hospitals in the last week of life. The word 'hospital' shares a common linguistic root with words like hospice and hospitality. Hospitality -- understood as being welcomed and cared for with kindness and attentiveness -- is still what everyone seeks when they come to hospital, including patients and their families who are going through the journey of dying, death and bereavement. That is why the Hospice Friendly Hospitals Programme (2007-2012) commissioned this first ever national audit of end-of-life care in Irish hospitals.This report contributes to the growing practice within the Irish hospital system of auditing performance against standards in order to ensure that every aspect of its work meets, and even exceeds, the highest standards of care and excellence. Given that end-of-life care standards did not exist at the time the audit -- but have since been published as Quality Standards for End-of-Life Care in Hospitals1 -- it may be more appropriate to regard this report as a "pre-audit" or "baseline-audit". It is Government policy, since February 2009, to introduce a mandatorylicensing system whereby each hospital will only be allowed to practice if, on the basis of audited performance, it meets acceptable quality standards of service

Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca2+ release in permeabilized pancreatic acinar cells

Inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca2+ release induced with 0.18 μM IP3 is half maximally inhibited with approximately 5 μM decavanadate. Complete inhibition is achieved with around 20 μM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP3, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca2+ uptake into intracellular stores, does not influence IP3 induced Ca2+ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP3 to the same cellular preparations was investigated. We found that binding of IP3 to a high affinity receptor site (Kd approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 μM decavanadate, IP3 binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP3 with the same affinity (Kd approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP3 was displaced from these subcellular membranes with similar kinetics by unlabelled IP3 and decavanadate. The data suggest that the inhibitory action of decavanadate on IP3 induced Ca2+ release is a consequence of its effect on binding of IP3 to its receptor. EGTA, ethylene-glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PEG, polyethylene glycol; IP3, inositol 1,4,5-trisphosphate; MOPS, morpholinopropane sulfonic acid; HEPES, N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid; Tris, tris(hydroxymethyl)-aminomethan