Screening of the human tumor necrosis factor (TNF) gene and its receptor 1 (TNFR1) gene for DNA alterations and the subsequent investigation of these and an IL12p40 polymorphism for an association with paediatric tuberculosis

Includes bibliographical references (leaves 85-96).Infection with Mycobacterium tuberculosis is characterised by diverse outcomes; the majority of infected individuals remain well and yet others develop disease ranging from limited pulmonary tuberculosis to severe disseminated disease. The reasons for this diverse outcome are poorly understood, but host factors are thought to play an important role. In particular, a genetic component to susceptibility to tuberculosis has been proposed. An important clue was the description of a group of Maltese children with an unusual susceptibility to progressive non-tuberculous mycobacterial infections. These patients showed defective tumor necrosis factor (TNF) production in response to endotoxin and a failure to upregulate TNF production in response to interferon gamma as well as diminished interferon gamma production during T-cell proliferation. They were found to lack expression of the interferon gamma receptor ligand-binding chain (IFN-yR1) on their cell surfaces due to a single point substitution resulting in a truncated protein. Since then other defects in the type 1 cytokine pathway leading to susceptibility to non-tuberculous mycobacteria, as well as to tuberculosis, have been described in rare isolated cases. From these findings, the hypothesis arose that less severe mutations in such pathways might individually, or in combination, lead to increased susceptibility to tuberculosis in the general population. The following study forms part of a larger multi-centre collaboration, which aims to better understand the genetic basis of susceptibility to mycobacterial infection by addressing this hypothesis. The approach taken has been the recruitment and immuno-phenotyping of a large group of children with tuberculosis as well as control subjects. Candidate genes, of the type 1 cytokine pathways being investigated, include interferon gamma, interleukin­12 and their receptors and TNF. The focus of the study described in this thesis has been the screening of a sub-cohort of patients and control subjects for DNA sequence alterations in the TNF and TNFR1 genes. The individuals in this cohort were selected on the basis of their whole blood stimulation assays, where either high or low levels of TNF in response to non-specific stimulatory factors, were the determining criteria. It was assumed that these two phenotypic groupings would be enriched for gene variants contributing to the TNF responses recorded in the stimulation assays. Once identified, these polymorphisms would be screened for frequencies in the broader patient and control groupings and assessed for any association with susceptibility to tuberculosis. This study was considered important in attempting to explain which genes and their polymorphisms are involved in determining the high prevalence of tuberculosis in African populations

The impact of vibrational Raman scattering of air on DOAS measurements of atmospheric trace gases

In remote sensing applications, such as differential optical absorption spectroscopy (DOAS), atmospheric scattering processes need to be considered. After inelastic scattering on N2 and O2 molecules, the scattered photons occur as additional intensity at a different wavelength, effectively leading to filling-in of both solar Fraunhofer lines and absorptions of atmospheric constituents. Measured spectra in passive DOAS applications are typically corrected for rotational Raman scattering (RRS), also called Ring effect, which represents the main contribution to inelastic scattering. In contrast to that, vibrational Raman scattering (VRS) of N2 and O2 has often been thought to be negligible, but also contributes. Consequences of VRS are red-shifted Fraunhofer structures in scattered light spectra and filling-in of Fraunhofer lines, additional to RRS. We describe how to calculate VRS correction spectra in analogy to the Ring spectrum. We discuss further the impact of VRS cross-sections for O2 and N2 on passive DOAS measurements. The relevance of VRS is shown for the first time in spectral evaluations of Multi-Axis DOAS data. This measurement data yields in agreement with calculated scattering cross-sections, that the observed VRS cross-section amounts to 2.2 ± 0.4% of the cross-section of RRS under tropospheric conditions. It is concluded, that this phenomenon has to be included in the spectral evaluation of weak absorbers as it reduces the measurement error significantly and can cause apparent differential optical depth of up to 2.5 × 10−4. Its influence on the spectral retrieval of IO, Glyoxal, water vapour and NO2 in the blue wavelength range is evaluated. For measurements with a large Ring signal a significant and systematic bias of NO2 dSCDs up to (−3.8 ± 0.4) × 1014 molec cm−2 at low elevation angles is observed if this effect is not considered

Red Queen competitive imitation in the U.K. mobile phone industry

This paper uses Red Queen competition theory to examine competitive imitation. We conceptualize imitative actions by a focal firm and its rivals along two dimensions: imitation scope, which describes the extent to which a firm imitates a wide range (as opposed to a narrow range) of new product technologies introduced by rivals; and imitation speed, namely the pace at which it imitates these technologies. We argue that focal firm imitation scope and imitation speed drive performance, as well as imitation scope and speed decisions by rivals, which in turn influence focal firm performance. We also argue that the impact of this self-reinforcing Red Queen process on firms’ actions and performance is contingent on levels of product technology heterogeneity—defined as the extent to which the industry has multiple designs, resulting in product variety. We test our hypotheses using imitative actions by mobile phone vendors and their sales performance in the U.K. from 1997 to 2008

Pathogenetische Eigenschaften von ERj3, ERj6 und Sec62

Ziel dieser Arbeit war die Charakterisierung der Hsp40-Cochaperone ERj3 und ERj6 sowie Sec62 in Bezug auf deren mannigfaltige Funktionen im endoplasmatischen Retikulum. Besondere Beachtung fanden hier die Auswirkungen des erhöhten Calciumausstroms über Sec61 nach Depletion jedes der genannten Proteine, namentlich dessen Effekte auf Migration, Proliferation und das Zytoskelett. Weiterhin wurden der Zusammenhang eines ERj6-Mangels mit Neurodegeneration und Diabetes mellitus untersucht. Eine letzte Zielsetzung fand sich in der Betrachtung des Interaktoms von Sec62 mit besonderem Blick auf die molekularonkologisch relevanten Interaktionspartner. Zur Klärung der gestellten Fragen kamen unterschiedlichste Methoden der Zellkultur und der Proteinbiochemie zum Einsatz. So zeigten HeLa-Zellen nach Depletion von ERj3 beziehungsweise ERj6 in Migrationsassays ein signifikantes Migrationsdefizit. Parallel mithilfe des xCelligence-Systems angefertigte Proliferationsanalysen legten dar, dass dieses unabhängig einer Vitalitätseinschränkung der untersuchten Zellen bestand. Um die Vulnerabilität ERj3- beziehungsweise ERj6-depletierter Zellen gegenüber ER-Stress zu evaluieren, wurden diese während der besagten Proliferationsanalysen mit Thapsigargin behandelt. Dies führte zu einem tendenziell erkennbaren, statistisch allerdings nicht signifikanten Proliferationsdefizit von HeLa-Zellen, in denen jeweils eines der Hsp40- Cochaperone RNA-Interferenz-basiert depletiert wurde. Ähnliches zeigte sich in fluoreszenzmikroskopischen Versuchen zum Aktinzytoskelett von HeLa-Zellen, das sich weder nach „silencing“ von ERj3 noch von ERj6 erkennbar veränderte. Die Auswirkungen eines konstitutiven ERj6-Mangels wurden anhand der Fibroblasten einer Familie mit erblicher Neurodegeneration und Diabetes mellitus untersucht. Diese zeigten in Western Blot-Kontrollen des UPR-induzierten Proteins GRP170 keinen dauerhaft erhöhten ER-Stress, reagierten jedoch tendenziell sensibler auf durch Tunicamycin induzierten ERStress als gesunde Kontroll-Fibroblasten. Die genannten Befunde sprechen zusammenfassend für Redundanzen in den Funktionen von ERj3 und ERj6, die sich durch zuvor publizierte Befunde zum additiven Effekt einer Co-Depletion auf das Calciumleck des ER bekräftigen lassen. Im Verlauf der beschriebenen Untersuchungen wurde eine Methode zur zuvor nicht möglichen Herstellung von Migrationsassays nach Plasmidtransfektion erarbeitet. Um das Interaktom von Sec62 weitergehend zu bestimmen, wurden Co- Immunopräzipitationen mit anschließender Western Blot-Kontrolle der präzipitierten Proteine durchgeführt. Hierbei wurde gezeigt, dass Sec62 mit Vimentin und Hsp70 interagierte. Beide Proteine wurden in der rezent veröffentlichten Literatur als potentielle Tumormarker oder Zielstrukturen einer „targeted therapy“ diskutiert. In der vorliegenden Arbeit wurden demnach die Hsp40-Cochaperone ERj3 und ERj6 als wichtige Komponenten in der Calciumhomöostase des endoplasmatischen Retikulums bestätigt und in ihren pathophysiologischen Eigenschaften näher charakterisiert. Gerade ERj6 zeigte sich hier als Bindeglied zwischen ER-Stress und Neurodegeneration sowie Diabetes mellitus. Sec62 deutete daneben als in der Tumorbiologie hochrelevante Struktur weitere Mechanismen seiner Einflussnahme auf die Entstehung maligner Erkrankungen an.The purpose of this work was the characterization of the hsp40-co-chaperones ERj3 and ERj6, as well as Sec62 in regard to their many functions in the endoplasmic reticulum. Special attention was payed to the effects of an increased calcium leakage from Sec61 after depletion of each of the aforementioned proteins, namely on its effects on migration, proliferation and the cytoskeleton. Another aim was the evaluation of a genetically caused loss of ERj6 and the resulting phenotypes of neurodegeneration and diabetes mellitus. Lastly efforts were undertaken to study the interactome of Sec62 which was done with a focus on proteins with relevance to molecular oncology. To answer the asked questions, different methods of cell culture and protein biochemistry were applied. For one, HeLa cells showed signifanct migratory impairment in migration assays after ERj3 or ERj6 were depleted. Simultaneously conducted proliferation analysis with the xCelligence system confirmed that this migratory impairment was not due to lack of vitality, but resulted from specific effects of the protein depletion. To evaluate the potential increase in vulnerability to ER stress, ERj3 or ERj6 depleted HeLa cells were treated with increasing amounts of thapsigargin during the xCelligence proliferation analysis. The observed proliferation deficit and increased susceptibility to thapsigargin in these cells showed tendencies of significance but did not hold up to statistic testing. Similarly, the RNA interference based silencing of one of the mentioned hsp40-co-chaperones did not lead to any changes in the structure of HeLa cells‘ actin cytoskeleton which was tested via immunostaining fluorescence microscopy. The consequences of a constitutive lack of ERj6 were studied by using cultured fibroblasts derived from a family with a history of inherited neurodegeneration and diabetes mellitus. These showed no signs of elevated basal ER stress. This conclusion was drawn after western blot analysis showed no difference in the GRP170 content of fibroblasts carrying the mutated ERj6 in comparison to healthy controls. However, the diseased cells reacted more sensitively to tunicamycin induced ER stress. All these findings support the theory that ERj3 and ERj6 share mutual functionalities and are redundant in regard to their functions in chaperoning and ER calcium homeostasis. This is further underlined by previously published data showing an additive effect on calcium leakage from the ER after co-silencing of the two hsp40-co-chaperones. While conducting research on ERj3, ERj6, Sec62 and their effects on the migratory potential of HeLa cells, a new method was established, allowing the hitherto impossible use of plasmid transfected cells in migration assays. To create a better understanding of the interactome of Sec62 co-immunoprecipitations were performed. After western blot analysis of the precipitated proteins, Hsp70 and Vimentin were shown to interact with Sec62. In recently published literature, both proteins were discussed as potential tumor markers or targets for personalized treatments. In this work, the hsp40-co-chaperones ERj3 and ERj6 were further characterized as important components of ER calcium homeostasis and their roles in pathophysiological processes were illuminated. Especially ERj6 turned out to be a link between ER stress, neurodegeneration and diabetes mellitus. Sec62 on the other hand cemented its standing in molecular oncology as a driving force in tumorigenesis and the development of targeted therapies

Measurements of reactive trace gases in the marine boundary layer using novel DOAS methods

Reactive Halogen Species (RHS) can have a large impact on tropospheric chemistry. Already small concentrations in the marine boundary layer (MBL) can have a significant impact on the global budget of ozone and other trace-gases. Shipborne measurements of BrO and IO were conducted in the MBL from 70°S to 90°N to obtain a global picture of their distribution over several years. A focus were upwelling regions, which can provide large amounts of precursor substances for RHS in the MBL, of which some were measured simultaneously by project partners. Measurements in the tropical Atlantic showed BrO concentrations below 2 ppt in agreement with previous observations. IO was detected with concentrations of up to 1 ppt, its distribution was found to correlate with surface water iodide concentrations. In the Peruvian upwelling region MAX-DOAS and CE-DOAS measurements showed agreeing surface VMR of up to 1.4 ppt. The MAX-DOAS retrieval agreed with previous satellite observations of IO in this region. For polar measurements, background concentrations of IO of 0.2-0.4 ppt during the respective summer period were observed. An additional source of reactive bromine in the MBL was observed for the first time from the ground: Polar air-masses were observed south of the Azores yielding BrO concentrations of 4-6 ppt. Glyoxal was not found in the remote MBL of the tropical Atlantic and Pacific above a detection limit of 35 ppt. Relative water vapour absorption band strengths in the wavelength interval from 400-480 nm were analysed systematically and correction factors from 0.5-2 were found, leading to improvements of the spectral retrieval. Vibrational Raman scattering of N2 and O2 was quantified for the first time in MAX-DOAS measurements, yielding a reduction of up to 30% of IO measurement errors

Retrieval interval mapping: a tool to visualize the impact of the spectral retrieval range on differential optical absorption spectroscopy evaluations

Remote sensing via differential optical absorption spectroscopy (DOAS) has become a standard technique to identify and quantify trace gases in the atmosphere. Due to the wide range of measurement conditions, atmospheric compositions and instruments used, a specific challenge of a DOAS retrieval is to optimize the retrieval parameters for each specific case and particular trace gas of interest. Of these parameters, the retrieval wavelength range is one of the most important ones. Although for many trace gases the overall dependence of common DOAS retrieval on the evaluation wavelength interval is known, a systematic approach for finding the optimal retrieval wavelength range and quantitative assessment is missing. Here we present a novel tool to visualize the effect of different evaluation wavelength ranges. It is based on mapping retrieved column densities in the retrieval wavelength space and thus visualizing the consequences of different choices of spectral retrieval ranges caused by slightly erroneous absorption cross sections, cross correlations and instrumental features. Based on the information gathered, an optimal retrieval wavelength range may be determined systematically

New methods for the calibration of optical resonators : integrated calibration by means of optical modulation (ICOM) and narrow-band cavity ring-down (NB-CRD)

Optical resonators are used in spectroscopic measurements of atmospheric trace gases to establish long optical path lengths L with enhanced absorption in compact in-struments. In cavity-enhanced broad-band methods, the ex-act knowledge of both the magnitude of L and its spectral dependency on the wavelength lambda is fundamental for the correct retrieval of trace gas concentrations. L(lambda) is connected to the spectral mirror reflectivity R (lambda), which is often referred to instead. L(lambda) is also influenced by other quantities like broad-band absorbers or alignment of the optical resonator. The established calibration techniques to determine L(lambda), e.g. introducing gases with known optical properties or measuring the ring-down time, all have limitations: limited spectral resolution, insufficient absolute accuracy and precision, inconvenience for field deployment, or high cost of implementation. Here, we present two new methods that aim to overcome these limitations: (1) the narrow-band cavity ring-down (NB-CRD) method uses cavity ring-down spectroscopy and a tunable filter to retrieve spectrally resolved path lengths L(lambda); (2) integrated calibration by means of op-tical modulation (ICOM) allows the determination of the op-tical path length at the spectrometer resolution with high ac-curacy in a relatively simple setup. In a prototype setup we demonstrate the high accuracy and precision of the new approaches. The methods facilitate and improve the determination of L(lambda), thereby simplifying the use of cavity-enhanced absorption spectroscopy.Peer reviewe

On the relative absorption strengths of water vapour in the blue wavelength range

In recent updates of the HITRAN water vapour H2O spectroscopic compilation covering the blue spectral region (here: 394–480 nm) significant changes for the absorption bands at 416 and 426 nm were reported. In order to investigate the consistency of the different cross-sections calculated from these compilations, H2O vapour column density ratios for different spectral intervals were retrieved from long-path and multi-axis differential optical absorption spectroscopy (DOAS) measurements. We observed a significant improvement of the DOAS evaluation when using the updated HITRAN water vapour absorption cross-sections for the calculation of the reference spectra. In particular the magnitudes of the residual spectra as well as the fit errors were reduced. However, we also found that the best match between measurement and model is reached when the absorption cross-section of groups of lines are scaled by factors ranging from 0.5 to 1.9, suggesting that the HITRAN water vapour absorption compilation still needs significant corrections. For this spectral region we present correction factors for HITRAN 2009, HITRAN 2012, HITEMP and BT2 derived from field measurements. Additionally, upper limits for water vapour absorption in the UV-A range from 330 to 390 nm are given