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Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on secretome analysis, Bacillus pumilus strain Jo2, possessing high secretion capability, was chosen for an omics based investigation. The physiology of Bacillus pumilus cells growing either in minimal or complex medium was analyzed by a combination of proteomic and metabolomic methods. Master gels of the cytosolic and the secreted proteome covering major parts of the main metabolic pathways were created by means of 2D gel electrophoresis. Quantification of 2D gels allowed displaying the most abundant proteins in these sub-proteomes. Application of the GeLC-MS/MS technique tripled the number of identified proteins and enabled detection of many intrinsic membrane proteins. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43 % of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H-NMR methods numerous metabolites were analyzed and assigned to the reconstructed metabolic pathways. Our data indicate that applying a combination of proteomic and metabolomic techniques a comprehensive view of the physiology of growing B. pumilus cells can be gained. In addition, selected production-relevant genome features such as the restriction modification system, NRPS clusters and the secretory system of B. pumilus Jo2 are discussed. In their natural habitat, the soil, B. pumilus cells are often exposed to growth limiting conditions due to the lack of sufficient amounts of nutrients. Such limitations can also occur during fermentation conditions and will negatively influence the efficiency of the process. Glucose is the main carbon and energy source of B. pumilus. Thus, a deficiency of glucose has an enormous impact on cell growth. A 1D LC-MS/MS approach was performed to quantify the proteins using an N14/N15 labeling and to analyze the changes in the protein equipment when B. pumilus cells stop their exponential growth and become stationary due to limitation of glucose. 1033 proteins in the cytosolic fraction of B. pumilus cells were quantified and 272 of them appeared to be upregulated when the cells experience glucose starvation. 2D-PAGE was used to analyze the exoproteome of those cells. Glucose starving B. pumilus cells seemed to focus on usage of proteins and peptides as alternative carbon and energy sources instead of other carbohydrates. Especially the exoproteome of glucose starving cells is dominated by proteases and peptidases. Furthermore, cells used fatty acids as carbon source indicated by upregulation of enzymes involved in β-oxidation and the methylcitrate pathway. Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus. Using the physiological knowledge gained during our studies, we analyzed samples taken during an industrial fermentation process. Five samples were taken during the processes using a protease overexpressing B. pumilus strain and a non-overexpressing B. pumilus reference strain. 2D-PAGE was employed to analyze the samples. 448 proteins could be identified in the samples from the protease overexpressing stain as well as 453 proteins in the reference strain. The proteins were quantified relatively comparing the different growth phases of each strain as well as comparing the strains to each other. The physiological knowledge gained from the shake flask studies enabled us to interpret the findings. Both strains showed an induction of proteins involved in acquisition of alternative carbon sources and of proteins involved in degradation and usage of fatty acids, e.g. the methylcitrate pathway, when they stop exponential growth. This is comparable to the results gained from the analysis of B. pumilus cells under glucose limitation, indicating similar conditions during the processes. Especially in the late phases of the fermentation processes the cells were obviously exposed to severe stress conditions. Our results demonstrated that overexpressing cells showed a significantly stronger oxidative stress response at the end of the fermentation process compared to non-overexpressing cells, which indicated that not only the high cell densities but also the overproduction of the target protein might be responsible for these conditions.
All types of muscles use Ca2+ as their main intracellular messenger. In skeletal muscle fibers abnormal levels of intracellular calcium result in altered contractile properties, altered energy metabolism, and altered gene expression. Moreover, long term failure of normal Ca2+ homeostasis can lead to cell death of muscle fibers by necrosis and apoptosis. Elevations of intracellular Ca2+ levels are more and more regarded as the reason for pathological changes and muscle fiber damage in Duchenne Muscular Dystrophy (DMD). DMD is a severe recessive x-linked muscle disease caused by mutations in the dystrophin gene. The characteristics of DMD are muscle tissue wasting and fibrosis. Both muscle wasting and intracellular Ca2+ are to be reflected in changes of muscle force. Several Ca2+ conducting channels including transient receptor potential (TRP) channels are supposed to account for the abnormal Ca2+ homeostasis in DMD. Gene expressions of TRP channels have been studied in human and mouse skeletal muscle and among others TRPC3, TRPC6 and TRPV4 channels were found to occur in skeletal muscles. The present study followed the hypothesis that TRPC3, TRPC6 and TRPV4 are functional in skeletal muscle fibers and that they contribute to muscular Ca2+ homeostasis. Further, it was assumed that dysfunction of the mentioned TRP channels contributes to abnormal contractile properties and pathology and of dystrophin-deficient muscle. To study Ca2+ changes in mouse skeletal muscle fibers the fluorescent calcium indicator Fura-2 was used. Further, the technique of Mn2+ quench of Fura-2 fluorescence was applied. Muscle force measurements of mouse soleus and diaphragm strips were performed. To elucidate abnormalities of TRP channel function in dystrophin-deficient muscle, muscles and muscle fibers of mdx mice were studied. Hyperforin, an activator of TRPC6 channels elicited increases of calcium levels in wildtype muscle fibers. These increases were partly inhibited by the TRPC6 inhibitor 1-(5-chloronaphthalenesulfonyl) homopiperazine hydrochloride (ML-9). The TRPC3/TPRC6 activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in increased calcium entry, which was attenuated by ML-9. 2-aminoethoxydiphenylborane (2-APB), an unspecific TRP channel inhibitor, suppressed calcium entry in muscle fibers under basal conditions. In addition, the specific TRPC3 inhibitor Pyr3, strongly inhibited background calcium entry. The TRPV4 activator 4α-phorbol 12,13-didecanoate (4α-PDD) induced significant increased calcium entry and this increase could be inhibited by the TRPV4 inhibitor HC 067047. During muscle force recordings ML-9 significantly inhibited twitches and tetani and accelerated muscle fatigue during sustained repetitive stimulation. The results indicate that TRPC3, TRPC6 and TRPV4 are functionally expressed in mouse muscle fibers. TRPC3 stays active under the basal conditions and contributes to background calcium entry. In contrast, TRPC6 and TRPV4 did not seem to be active at resting conditions, but could be pharmacologically activated. TRPC6 may play a role to counteract the calcium loss under long-term muscle fatigue. Though TRPC3 and C6 play a role for muscular Ca2+ homeostasis, it is unclear whether and how the two channels associate and cross-talk with each other in skeletal muscle cells. In mdx fibers Pyr3 inhibited background calcium influx stronger that in WT fibers, implying a possible over-activation of TRPC3 channels in mdx muscle fibers. At later stages mdx muscle showed marked decrease in force reflecting muscle wasting. Soleus showed moderate decrease and diaphragm showed severe decrease (more than 60%) in force. Resistance to muscle fatigue was shown in mdx soleus muscle when compared with WT soleus muscle. Diaphragm segments of mdx mice showed very strong resistance to muscle fatigue. The results indicate a substantial loss of muscle mass, an increase in oxidative fiber types and a reduction of fast fatigable muscle fibers. It is concluded that the hypothesis of functional expression of TRPC3, TRPC6 and TRPV4 in mouse skeletal muscle has been confirmed. The results give improved knowledge about the relation of Ca2+ homeostasis, mdx pathology and TRP channels. Diaphragms of old mdx mice show severe muscle weakness but the remaining fibers of the diaphragm showed strong fatigue-resistance. The application of a TRPC3 inhibitor may be a promising treatment to prevent high Ca2+ mediated muscle damage in muscular dystrophy.
Background: Despite of the remarkable caries reduction in permanent dentition, caries levels of primary teeth has stagnated in Germany. Early Childhood Caries (ECC) or also known as baby bottle tooth decay is the most vulnerable form of caries in young children, but minimal data and information from different German states are available to determine the appropriate preventive programs. Aim: The purpose of the current study is to find the prevalence of ECC among young children in the state of Mecklenburg-Vorpommern (North-East Germany) and to optimize an intervention on ECC prevention in a community setting. In addition to education, fluoride varnish is evaluated on young children with active ECC. Design: In this cross-sectional study, a total of 4283 children living in the state of Mecklenburg-Vorpommern were examined. Four age groups - with an accuracy of one day - were formed as follows: less than one year (n=8), one year (n=293), two years (n=1618) and three years (n=1888). The examination was carried out by community dental service’s examiners whom are calibrated to ECC diagnostic criteria of Robke and Buitkamp (2002), and dmf-t values for caries diagnosis. These data are compared by those of children (n=5355) of same age group for the year 2011-2012. In addition, a structured questionnaire on the starting preventive programme on ECC was filled out by the community dentists and for the city of Greifswald, fluoride varnish (Duraphat®, 5% NaF = 2.26%F, Colgate-Palmolive, Germany) was applied for 32 children previously diagnosed with active ECC (ECC1: n=15, ECC2: n=17). Lesions are identified as active or non active according to texture and luminosity, and oral hygiene index (OHI-S) is measured and re-evaluated at three months follow up. Results: The percentage of children under three years old in 2012-2013 with ECC was comparatively low (4%) which possibly reflects the very young age of the children and a restriction for ECC on the upper incisors. The overall caries prevalence in Mecklenburg-Vorpommern varied from 9% to 15%. Most cavitated lesions are untreated. These results are comparable with the results from other German counties. The interventions of the ECC programme vary considerably among the different counties. There was no significant difference in the oral hygiene index (OHI-S) prior and post fluoride varnish application (p-value = 0.25). The use of fluoride varnish resulted in an 81%, statistically significant decrease of active ECC lesions in Greifswald (p < 0.001). Conclusion: The prevalence of caries among young children was considerable in Mecklenburg-Vorpommern. A preventive intervention in nurseries and fluoride varnish applications for active ECC lesions seems to be a feasible approach in controlling caries in early childhood. However, further quality management and standardization of the program should be reinforced.
Decades after international guidelines to approach Universal Health Coverage and Access for All to essential health care services have been formulated by the global community, social protection in health remains a major global challenge. This implies the devastating situation of having less than 15% of the global population benefiting of any kind of social protection in health, while more than 70% of the world population lacks any type of social protection coverage. 36 years after the famous and often-cited Alma-Ata Declaration proclaimed that „the promotion and protection of the health of the people is essential to sustained economic and social development and contributes to a better quality of life and to world peace”, people of the informal sector – which forms up to 90% of the population in many countries of sub-Saharan Africa – are still forced to take out loans or sell their assets to settle their hospital bills and in the end fall into poverty because of unbearable health care costs. While private health insurance schemes are mainly serving people living in urban areas and offer products and services that are not tailored to the needs of people of low-income from rural and/or remote areas, public social health insurance schemes are usually designed to serve the formal sector or are exclusively catering for public servants. At the same time, social protection in health is increasingly regarded to be a guarantor for development and economic growth of the national economy. In this context, some authors are convinced that community-based health financing is to be seen as a promising approach to insure parts of the population, which are normally excluded from any type of social protection in health, against catastrophic health care costs. With a focus on low-income people, Community-based Health Financing (CBHF) schemes offer products, processes and institutions that are tailored to the specific needs of their low-income target group, usually situated in the informal sector. In the aim to meet international standards and comply with the global development agenda, governments in sub-Saharan Africa are increasingly acknowledging the need to include the informal sector and people of low-income into their public health financing systems. As a result, innovative health systems evolved, which often comprise of hybrid sub-systems to cover various target groups of the society. While some governments – such as the governments of Rwanda, Ghana and Tanzania – have already implemented integrated national Social Health Insurance (SHI) systems that consider CBHF schemes to cover the informal sector, others are aiming at implementing this innovative idea in the near future, e.g. Burkina Faso and Togo. Given the above-illustrated situation, the overall research objective of this thesis is to explore the potential contribution of CBHF schemes towards Universal Health Coverage (UHC) in low- income countries of sub-Saharan Africa. Furthermore, the specific research objectives are set as follows; (1) To establish common lessons learnt from low-income countries in sub-Saharan Africa which implemented integrative SHI systems by combining efforts of national SHI schemes and CBHF schemes, or which are in an advanced stage of designing and implementing the same. (2) To comprehensively analyze the Kenyan health financing system and design adequate interventions towards the design and implementation of an integrative national SHI scheme in Kenya which is favoring UHC. (3) To develop a standard model for implementing integrative SHI systems in low-income countries of sub-Saharan Africa and the world. This thesis will at first provide a comprehensive topical background containing evidence about different relevant concepts such as Development, Universal Health Coverage, Social Protection, Health Financing and Micro Health Insurance. On this basis, the potential of combining community-based and national efforts towards tailored health care financing at national level will be explored by analyzing strengths and weaknesses of both approaches and providing brief insights from low-income countries of sub-Sahara Africa in this area. Furthermore, a comprehensive background to common development initiatives as well as the social protection and health care financing sectors in Kenya is provided to introduce the case study of chapter four. In the third chapter, common efforts of governments and other stakeholders involved in health care financing in sub-Saharan African countries to integrate CBHI schemes into public SHI schemes will be reviewed and analyzed. In the scope of this review, Tanzania, Rwanda, Burkina Faso and Ghana will serve as practical country case examples. Based on this extensive cross-country analysis, common lessons learnt regarding the complex process of designing integrative SHI systems in low-income countries of sub-Saharan Africa will be presented. In chapter four, through a comprehensive country case study, the Kenyan health and health financing sector and its stakeholders will be analyzed regarding its potential towards UHC, aiming at the development of most promising interventions towards the design and implementation of an integrated SHI scheme in Kenya, considering CBHF schemes as one building block of the system. A multi-stage model as well as a multi-level structure of a national SHI system to approach UHC in Kenya will be outlined and presented. The thesis will be concluded in chapter five by transferring the Kenyan experience to a global level and suggesting a standard model for implementing integrated SHI schemes in similar contexts as given in Kenya and the presented case examples. In the conclusion, common opportunities and limitations of community-based approaches towards UHC are highlighted and a way forward for the Kenyan context is suggested.
The focus of this study is on the geochronological and paleo-climatic characterization of late Pleistocene glaciations in Turgen and the Khangai Mountains located in central and western Mongolia. These two mountain ranges form a 700 km long NW-SE transect through Mongolia and allow assumptions of the temporal and causal dynamics of the regional late Quaternary glaciations and their correlation to other mountain glacier records from Central and High Asia. In order to evaluate extent and timing of the Pleistocene glaciations in Mongolia, geomorphological mapping and cosmogenic radionuclide (CRN) surface exposure dating (10Be) were carried out in four valley systems located in the Khangai and Turgen Mountains. Additionally, a coupled 2-D surface energy balance and ice flow model was used to determine steady-state conditions for glaciers under various climatic scenarios. With this model it is possible to test combinations of temperature and precipitation settings, which would produce glacier configurations that fit the field-mapped ice extent. In total, 47 glacial boulders and roche moutonnées were sampled, prepared and AMS measured to determine the absolute timing of moraine formation and ice retreat based on 10Be surface exposure dating. Of these, 27 samples were obtained from the Khangai Mountains (three separate moraine sequences) and 20 samples were taken from the Turgen Mountains (two moraine sequences). The dating results (presented as minimum ages) give evidence for a late Pleistocene maximum ice expansion during late MIS 5 (81−78 ka) and major ice advances during MIS 2 (26−20 ka) in both mountain ranges. Only in the Khangai Mountains (central Mongolia) very significant glacier advances also occurred during mid-MIS 3 (49−35 ka), which exceeded the ice limits set during the MIS 2 glaciation. A final ice position, constructed shortly before the onset of full ice retreat was formed between 19-16 ka, and is likely to represent a recessional ice stillstand, or alternatively a final ice readvance during the early part of the last-glacial-interglacial-transition (LGIT) in both mountain ranges. Energy/mass balance and ice flow modeling results suggest that climatic conditions during the MIS 5 and MIS 3 maximum advances in the Khangai Mountains were depressed between a ∆T of -6.0 to -5.2 °C with a precipitation factor of 1.25-1.75 (P = 125-175 %, compared to modern conditions), and a ∆T of -5.3 to -4.4 °C (P = 75-125 %), respectively. For the MIS 2 ice advances modeling results from the Turgen and Khangai Mountains suggest a temperature depression ∆T of -5.7 to -4.6 °C (at 22 ka; P = 25-50 %) in the East-Turgen, and a ∆T of -7.5 to -6.6 °C (at 20 ka; P = 25-50 %) in the Chulut area (Khangai Mountains). These results document a 1.8 - 2 °C difference of the modeled temperatures required to expand the studied paleo-glaciers in the Turgen and Khangai mountains to their field-mapped MIS 2 ice limits, highlighting a spatially differentiated pattern of paleo-temperature lowering across the studied 700 km NW-SE transect. Taken together, the presented record indicates that the largest ice advance in both investigated mountain ranges occurred during the MIS 5 / MIS 4 transition, despite earlier suggestions by previous studies that the local glacial maximum would be associated with the coldest periods of the last glacial cycle (i.e. MIS 4 or MIS 2). Glacier systems in the Khangai Mountains also increased substantially during MIS 3 (local LGM) in response to cool but comparable wet conditions, probably with a greater-than-today input from winter precipitation and an additional input of recycled moisture from expanded paleo-lakes in the Valley of the Great Lakes. The lack of a severe cooling during the MIS 3 ice advances, and probably also during the late MIS 5 ice expansion, suggests that variations in atmospheric circulation patterns, with its significance for controlling the regional precipitation/moisture supply, was a key driver for these late Pleistocene ice advances in Mongolia. This notwithstanding, there is also clear evidence for the development of an extensive glaciation during MIS 2, coinciding with a period of severe cooling and hyperarid conditions. This highlights that glacier systems in Mongolia responded sensitively, both, to variations in moisture supply and its seasonal distribution, and to the marked insolation minima during the last glacial cycle.
We consider Iterated Function Systems (IFS) on the real line and on the complex plane. Every IFS defines a self-similar measure supported on a self-similar set. We study the transfer operator (which acts on the space of continuous functions on the self-similar set) and the Hutchinson operator (which acts on the space of Borel regular measures on the self-similar set). We show that the transfer operator has an infinitely countable set of polynomial eigenfunctions. These eigenfunctions can be regarded as generalized Bernoulli polynomials. The polynomial eigenfuctions define a polynomial approximation of the self-similar measure. We also study the moments of the self-similar measure and give recursions for computing them. Further, we develop a numerical method based on Markov chains to study the spectrum of the Hutchinson and transfer operators. This method provides numerical approximations of the invariant measure for which we give error bounds in terms of the Wasserstein-distance. The standard example in this thesis is the parametric family of Bernoulli convolutions.
Background: Cardiovascular disease (CVD) remains the major cause of mortality and morbidity worldwide and produces large productivity loss. The majority of CVD mortality could be prevented with changes in modifiable risk factors including tobacco use, physical inactivity, unhealthy diet and harmful use of alcohol. Successful behavioral prevention of CVD requires the identification of relevant target behaviors and reach of populations at risk. Presenteeism i.e. attending work while ill is discussed as a work-related risk factor for CVD. However, little is known about the interplay of presenteeism with established health risk behaviors. The first aim of this dissertation was to examine the association of presenteeism with health behaviors (study 1). The second aim was to examine factors that can enhance the public health impact of CVD prevention efforts. Therefore, the effect of recruitment strategy used on reach (study 2) and of communication channel used on intervention usage (study 3) was examined. Methods: Study 1 comprised data from 710 Australian employees aged 18 years and older who completed an online-survey. Linear regression analysis was used to examine the association of health behaviors (physical activity, work and non-work-related sitting time, sleep duration and sleep quality) with presenteeism. For study 2 individuals aged 40-65 years were invited to a two-stage cardio-preventive program including an on-site health screening and a cardiovascular examination program (CEP) using face-to-face recruitment in general practices (n = 671) and job centers (n = 1,049), and mail invitations from a health insurance company (n = 894). Recruitment strategies were compared regarding three aspects of reach: (1) participation rate, (2) participants’ characteristics i.e. socio-demographics, self-reported health and CVD risk factors, and (3) predictors of program participation. Study 3 compromised 16,948 users (aged 18 years and older) of the feely available physical activity promotion program 10,000 Steps. Users were grouped based on which platform (website, app) they logged their physical activity: Web-only, App-only, or Web-and-app. Groups were compared on socio-demographics, engagement parameters and logged physical activity. Non-usage attrition i.e. discontinued program usage over the first three months was examined using Kaplan-Meier survival curves. A Cox regression model was used to determine predictors of non-usage attrition. Results: Analyses from study 1 revealed that presenteeism was associated with poor sleep quality and suboptimal sleep duration after controlling for socio-demographics, work and health-related variables. Engaging in three health risk behaviors was associated with higher presenteeism compared with engaging in none or one. Study 2 showed screening participation rates of 56.0%, 32.8%, 23.5% for general practices, job centers and the health insurance company, respectively. Participation rate for the CEP among eligible individuals was 80.3%, 65.5%, and 96.1%, respectively. Job center clients showed the lowest socio-economic status and the most adverse CVD risk pattern. Whereas being female predicted screening participation across all strategies, higher age predicted screening participation only within individuals recruited via the health insurance company. Within general practices and job centers CEP participants were less likely to be smokers than non-participants. Study 3 revealed that engagement with the program was highest for Web-and-app users. Cox regression showed that user group predicted non-usage attrition: Web-and-app users (hazard ratio = 0.86; P < .001) and App-only users (hazard ratio = 0.63; P < .001) showed a reduced attrition risk compared to Web-only users. Further, older age, being male, being non-Australian, higher program engagement and higher number of steps logged were associated with reduced non-usage attrition risk. Conclusion: The results of this dissertation have three implications for designing CVD behavioral interventions with a high public health impact. First, employees suffering from presenteeism may require interventions addressing health risk behaviors including suboptimal sleep behaviors. Second, implementing prevention efforts in job centers may be especially useful to reduce health inequalities induced by social gradient. Third, the population impact of web-based interventions may be increased when using mobile delivery channels.
In this Ph.D. project a method is developed to measure the magnetic field and to derive variations in the total plasma pressure due to (dia-) magnetic effects. For this purpose a plasma diagnostic has been set up at the fusion experiment ASDEX Upgrade to measure spectroscopically polarized light. The light is emitted from fast beam-particles excited by the plasma. Since the fast atoms travel through a magnetic field at high velocity, a strong Lorentz field is seen in the moving frame. This electric field gives rise to the so-called motional Stark-effect (MSE) and it is possible to conclude from the Stark-spectrum on the magnetic field.
Background: Microvascular Decompression represents an effective treatment for hemifacial spasm. The use of lateral spread responses (LSRs) monitoring remains a useful intraoperative tool to ensure adequate decompression of the facial nerve. Objective: To assess the value of LSRs intraoperative monitoring as a prognostic indicator for the outcome of microvascular decompression in hemifacial spasm. Methods: Our study included 100 patients prospectively. The patients were classified into 4 groups whether LSRs were totally, partially, not relieved or not detected from the start. According to clinical outcome, the patients were classified into 4 groups depending on the clinical course after surgery and the residual symptoms if any. Then, correlations were made between LSRs events and treatment outcome to detect its reliability as a prognostic indicator. Results: LSRs were relieved totally in 56% of the patients, partially relieved in 14%, not relieved in 10% and were not detected in 20% of the patients from the start. HFS was relieved directly after operation in 62% with clinical improvement of 90-100%. 31% described 50-90% improvement over the next 3 months after surgery. Almost all of these 31% (28 out of 31 patients) reported further clinical improvement of 90-100% within one year after surgery. 3% suffered from a relapse after a HFS-free period and 4% reported minimal or no improvement describing 0-50% of the preoperative state. The percentage of the satisfied patients with the clinical outcome who reported after one year a clinical improvement of 90-100% was 90%. Statistical analysis did not find a significant correlation between the relief of LSRs and clinical outcome. Conclusion: LSRs may only represent an intraoperative tool to guide for an adequate decompression but failed to represent a reliable prognostic indicator for treatment outcome.
This thesis draws a comprehensive picture about the radiation and diversification of truncatelloidean gastropods across the south pacific. It covers three more specifc studies focussing on the Truncelloideans from Fiji, Vanuatu and New Caledonia, respectively. And a conclusive analysis that combines the results of the three more specific studies and enhances them using species from the Austral Islands, Lord Howe Island, the Indonesian island Sulawesi as well as several species from New Zealand and Australia. Molecular phylogenies were calculated using four nuclear gene fragments (ITS2; 18S rRNA; 28S rRNA and Histone 3) besides the mitochondrial COI and 16S rRNA. Further molecuular data was used to calculate dated phylogenies, perform ancestral range reconstructions and develop a modified molecular barcoding approach.
Protamine is administered as protamine sulfate to reverse the anticoagulant effect of heparin following cardiopulmonary bypass surgery. Immunogenicity of protamine has been recognized for decades in several patient groups including vasectomized men, diabetic patients on protamine-containing insulin and patients undergoing cardiopulmonary bypass surgery. Anti-protamine/heparin antibodies are a newly described class of heparin-dependent antibodies found in about 30% of patients exposed to protamine and heparin during cardiac surgery. A subset of seropositive patients especially who tested positive for platelet-activating anti-protamine/heparin immunoglobulin G (IgG) antibodies before surgery have prolonged postoperative thrombocytopenia with an increased risk for arterial occlusions. Studies presented in this thesis shed light on potential approaches that may prevent antibody-mediated platelet activation by anti-protamine/heparin antibodies. Two approaches are presented in this thesis, partially desulfated heparin (ODSH) and low molecular weight protamine (LMWP). Our studies demonstrated the ability of ODSH to inhibit anti-protamine/heparin antibody-mediated platelet destruction in the NOD/SCID mouse model by: i) reduction of antibody binding to preformed protamine/heparin complexes, as shown by enzyme immunoassay, ii) interfering with the binding of protamine/heparin complexes to platelets as shown by flow cytometry and fluorescence microscopy, and iii) inhibition of antibody-mediated platelet activation. Interestingly, ODSH was also able to block ongoing platelet destruction by displacing pre-bound complexes from the platelet surface. In addition, our data suggest the use of synthesized LMWP as a substitute for protamine in heparin reversal. The in vitro investigations showed that synthesized LMWP efficiently neutralizes heparin using the activated partial thromboplastin time. Anti-protamine/heparin antibodies have low binding properties to LMWP/heparin complexes as indicated in enzyme immunoassay. The ability of platelet-activating anti-protamine/heparin antibodies to induce platelet activation in the functional assay was significantly reduced in the presence of LMWP/heparin compared to protamine/heparin complexes. Owing to findings obtained in our studies, both approaches might be a promising future option to reduce anti-protamine/heparin antibody-mediated adverse effects.
In this thesis, rates and extend as well as the ecological implications of electron exchange reactions that involve redox-active moieties in organic matter (OM) were explored. The research builds on earlier findings that confirmed that OM may act as terminal electron acceptor (TEA) for electrons released in microbial respiration. This property was associated with quinone moieties that are ubiquitously found in OM from terrestrial and aquatic environments and that may undergo reversible reduction to the respective hydroquinone. Earlier methodological advances allowed for a rapid, direct and precise quantification of the electron accepting and donating properties of quinones in dissolved OM (DOM) by mediated electrochemical analysis. In this work, the previously established mediated electrochemical analysis was adapted and used in the characterization of redox properties of particulate natural samples that contain redox active iron and organic matter ("geochemical phases"). For the first time, direct measurements confirmed that microorganisms transferred electrons (e) from microbial respiration to the organic and inorganic electron acceptors in the particulate phase. Particulate OM in the sediments was found to provide a capacity to accept or donate e of 650 µmol e/gC. An incubation experiment resolved the spatiotemporal dynamics of organic and inorganic TEA species (i.e., nitrate, sulfate, Fe- and Mn oxyhydroxides) in sediments upon changes in oxygen availability and hence redox conditions. Oxygen is consumed when the reduced species are oxidized and, by this means, re-generate their electron-accepting capacity. The use of mediated electrochemical analysis allowed for the quantification of the redox state of the geochemical phases during their reduction and re-oxidation. The electron fluxes initiated by the oxic re generation of the TEAs nitrate, sulfate, Fe(III), Mn(IV) and quinoid moieties in OM were therefore directly monitored instead of modeled from the species’ distribution profiles in interstitial waters. The cyclic reduction and re-oxidation of redox species exposed to oxygen fluctuations was suspected to be a critical component of many aquatic ecosystems. In stratified lakes, extended sediment volumes are exposed to oxygen only upon lake overturn. Lake oxygen budgets are therefore influenced by benthic redox processes. The combined field and laboratory study showed that lake overturn seasonally introduces a finite amount of oxygen to the hypolimnion and that about 50% of the subsequent sediment oxygen consumption is exclusively associated with the re-generation of TEA species. These species previously formed in the sediment when organic matter was microbially decomposed during anaerobia. While lake overturn can completely mix epi- and hypolimnetic waters, small-scaled dynamics in temperature and oxygen availability may confine discrete parts of the water column with oscillations in physicochemical conditions. In the studied lake, a transient thermocline cyclically introduces oxygen to hypoxic hyplimnetic waters close to the pelagic redox interface. In the lake, organic TEAs may represent an important component of the total pelagic electron acceptor capacity. Due to the rapid and reversible redox reactions of DOM, reduced organic TEAs are re-generated upon dislocation to oxic parts of the water column. Results show that diurnal fluctuations of oxycline depth shape a micro-environment selecting for microbial species that are released from TEA limitations by OM in oxidized state. Pelagic microbial communities subjected to the same amount of OM in different oxidation states differed by more than 50% after one day. This work substantiates earlier findings that suggested that OM may be an important TEA species in many aquatic and terrestrial ecosystems. OM reduction in microbial respiration was shown to directly affect critical system parameters as bacterial activity, oxygen budgets and aquatic biodiversity. Both the microbial reduction and subsequent abiotic oxidation of OM are sufficiently fast for relevant interaction with oxycline fluctuation on different timescales. Given that organic TEAs are cyclically regenerated, a significant share of ecosystem respiration could be linked to OM reduction. This thesis demonstrated the new and important role electron exchange reactions in OM-rich environments play and explored the mechanism of this previously neglected part of lake functioning. As of today, linking the chemistry of aquatic turnover processes with the microbiological and physical conditions at redox interfaces remains challenging. In conclusions, by providing several cases from aquatic environments, this thesis contributes to the mechanistic understanding of OM reduction in microbial respiration. The results prompt for further research regarding the competitive inhibition of other respiration pathways, including the reductive production of the potent greenhouse gas methane.
Background: Demographic changes are leading to a rapid increase in the number and proportion of the elderly. This goes along with an increase of prevalence of age-associated illnesses, such as dementia. The prevalence of dementia is estimated to amount to 1.5 million in Germany. Up to three-quarter of the persons with dementia (PWD) were living in their own homes. In European countries, dementia is associated with substantial and increasing healthcare costs, which makes dementia one of the most expensive diseases in old age and a serious health care priority. Whereas analyses of total healthcare costs in dementia have been the focus of various cost-of-illness (COI) studies, so far little is known about several cost categories in detail. Firstly, detailed economic analyses of medication cost are currently still missing. Secondly, it is well known that dementia is under-diagnosed, but there is a lack of knowledge about the differences in resource utilization and its costs between dementia patients with and those without a formal dementia diagnosis. Finally, analyses that take the utilization and costs of professional formal and unpaid informal care as well as caregiver’s productivity losses into a consideration are currently missing. Objectives: (1) To determine medication cost, cost per drug and number of drugs taken and analyze their associated factors; to estimate the current price reduction of anti-dementia drugs due to implementation of low-priced generics. (2) To determine health care resource utilisation and costs of patients with a formal diagnosis and those without a formal diagnosis of dementia, and to analyse the association between having received a formal dementia diagnosis and health care costs (3) To determine the utilization and costs of formal and informal care for PwD, indirect costs because of productivity losses of caregivers and the associations between cost, socio-demographic and clinical variables. Methods: The present study is a cross-sectional analysis of health care resource utilization and health care cost of community-dwelling PWD in primary care. Analyses are based on primary data from the ongoing DelpHi-MV trial (Dementia: Life- and person-centered help in Mecklenburg-Western Pomerania, Germany), a population-based, cluster-randomized, controlled intervention trial in the primary care setting (Clinical Trials gov. Identifier: NCT01401582). Eligible patients (older than 70 years, living at home) were screened in participating general practitioner practices for dementia using the DemTect. The utilization of healthcare resources was assessed within the baseline assessment at practitioner’s homes. Costs were calculated from the perspective of the statutory health insurance or the social perspective. Factors associated with healthcare cost were evaluated using multiple regression models. Results: (1) Medication cost and cost per drug were higher and the number of taken drugs lower in advanced stages of cognitive impairment. Prescription of anti-dementia generics could decrease overall medication cost by 28%. Medication cost was associated with number of diagnoses, deficits in activities of daily living and age. Dementia severity was related to cost per drug and number of drugs taken. (2) Patients formally diagnosed with dementia were treated significantly more often by a neurologist, but less often by all other outpatient specialists, and received anti-dementia drugs and day care more often. Diagnosed patients underwent shorter and less frequent planned in-hospital treatments. Dementia diagnosis was significantly associated with higher costs of anti-dementia drug treatment, but significantly associated with less total medical care costs, which valuated to be € 5,123 compared, to € 5,565 for undiagnosed patients. (3) Formal care were utilized less (26.3%) than informal care (85.1%), resulting in a cost ratio of one to ten (1,646 €; 16,473 €, respectively). In total, 29% of caregivers were employed, and every seventh (14.3%) experienced productivity losses, which corresponded to 1,258 € annually. Whereas increasing deficits in daily living activities were associated with higher formal and higher informal costs, living alone was significantly associated with higher formal care costs and the employment of a caregiver was associated with lower informal care costs. Conclusion: (1) Medication cost increases with the number of diagnoses and growing deficits in activities of daily living and decreases with age. Severely cognitively impaired persons are treated with a small number of high-priced drugs, which could suggest inadequate medication of multimorbid persons. (2) There are no significant differences in total health care cost between diagnosed and undiagnosed patients. Dementia diagnosis is beneficial for receiving cost-intensive anti-dementia drug treatments, but is currently insufficient to ensure adequate non-medication treatment for community-dwelling patients. (3) Informal care contributes the most to total care costs. Living alone is a major cost driver for formal costs because of the lower availability of potential informal care. The availability of informal care is limited and productivity losses are increased when a caregiver is employed.
The synthesis of valuable chemicals via traditional chemical methods can be often outperformed by the use of enzymes because of their excellent chemo-, regio- and stereoselectivity in aqueous solvents at ambient temperatures. On the other hand, enzymes often suffer from several limitations that hamper their industrial application. Protein engineering is commonly applied to overcome these limitations although the generation and the validation of mutants is often a laborious process that may not lead to the desired results within reasonable time frames. This thesis focuses on engineering the enantioselectivity and the substrate scope of industrially relevant enzymes, such as esterases and transaminases. Semi-rational protein engineering was employed to identify improved variants for the synthesis of valuable chemicals ensuring a reduced screening effort. Compared to previous works, 3DM’s applicability was extended to the study of correlated mutations and proved effective in the acceleration of the comprehension and in the mutation of these enzymatic scaffolds. Semi-rational approaches require an extensive amount of information such as protein structures, reaction mechanisms, previous mutational experiments reported in literature and a considerable amount of amino acid sequences from similar proteins to analyze amino acid distributions and correlated mutations. Here, we have exploited 3DM as a tool that can combine all this wealth of information: 3DM is a convenient solution to retrieve and integrate information simplifying decision making in the planning of a semi-rational mutant library since in 3DM’s multiple sequence alignments (MSA) is summarized Nature’s screening process for alternative variants. Furthermore, naturally evolving enzymes often require mutations at more than one position for the acquisition of a new property. Such mutations generate patterns that are recognized by the 3DM algorithm, which creates networks that can be investigated to design strategies that aim to improve the property of interest. Finally, these correlated mutations are connected to the mutations described in publications covered in the PubMed database, thus helping to investigate the role certain positions might play in the network. Article I shows that it is possible to improve the enantioselectivity of an esterase towards a highly symmetrical substrate while drastically reducing the screening effort. This was achieved through the creation of libraries that limit the variants to those identified in the 3DM alignment. Article II shows that networks of correlated mutations are composed of positions that may cluster around a function. These functions can be investigated because 3DM connects the positions in the network to their related publications. In this article, a mutant of the esterase PFE-I from Pseudomonas fluorescens was generated having increased enantioselectivity in the hydrolysis of important target compounds. Article III suggests that the in silico modelling software YASARA, combined with the use of the 3DM database, can further reduce the screening effort: it was possible to identify a hot-spot because both the 3DM database and YASARA docking studies, indicated its importance. This led to a further improved enantioselectivity of the enzyme variant identified in Article II. Article IV shows how MSA may be used to get structural insights into the catalytic properties of enzymes with documented activity. The study of the patterns observed in a large subfamily alignment allowed the definition of the structural determinants important for the substrate recognition in amine transaminases. Article V and VI apply the knowledge acquired for the improvement of the substrate scope in the amine transaminase from Vibrio fluvialis.
Interactions between bacteria and the human body are manifold and happen constantly. Most parts of the skin and gastrointestinal tract, the saliva, the oral mucosa, the conjunctiva and the vaginal mucosa are colonized with a multitude of bacterial species forming the human microbiota. Strikingly, the estimated amount of bacterial cells outnumbers the human body by 10 to 1. However, most of these bacteria colonize the human body without positive or negative effects and are regarded as commensals. Staphylococcus aureus a Gram positive bacterium is such a commensal bacterium of 25 % to 30 % of the world population. It is also an opportunistic pathogen and is able to cause infections in the lung, skin and heart and to induce sepsis. Its pathogenicity is mainly facilitated by the secretion of a broad spectrum of virulence factors which interact with the host. Some are distracting the immune system, others are targeting the host cell membrane or degrade macromolecular structures of the host in order to provide nutrients. Furthermore S. aureus is able to invade the host cell and to survive and replicate in the host cell cytosol or other compartments. The Gram negative proteobacterium Burkholderia pseudomallei is an environmental bacterium but still has the ability to enter the human body via body orifices or skin wounds. In a very efficient way it penetrates the host cell, replicates intracellular and the uses host structures to spread from cell to cell thereby causing the disease melioidosis often with fatal outcomes. Since the natural habitats of B. pseudomallei are wet soils, the change to the environment in the human body is drastic and requires a high degree of flexibility of the bacterium. Environmental stress conditions such as temperature, pH, nutrient limitation or presence of antibiotics induce a switch of colony morphology which is a special characteristic of this bacterium. Since it is assumed, that changes in colony morphology are connected to adaptive processes to the environmental changes, these morphology switches might also be important during infection. The host organism and the host cell on the other side try to kill and remove the bacterial threat by activating the immune system and cellular defence mechanisms. This includes generation of reactive oxygen and nitrogen species, production of antimicrobial peptides and cellular processes such as phagocytosis, autophagy, apoptosis and activation of the immune response. The actions and reactions on both, the pathogen side and the host side, are summarized as host-pathogen interactions. In the field of functional genomics, methods were developed to understand various levels of host-pathogen interactions. The holistic analysis of the mRNA (the transcriptome) or translated proteins (the proteome) were already very useful tools to describe important cellular processes on the host and the pathogen site. The level of metabolites with regard to host-pathogen interactions however, has been neglected so far. In this dissertation the metabolic composition in the intracellular and extracellular space of the host and the pathogen was analyzed. For this matter biochemical analytical tools were used such as 1H-nuclear magnetic resonance spectroscopy and chromatographic methods (GC and HPLC) coupled to mass spectrometry. The combination of these methods allows a broad coverage of physicochemical diverse metabolites. In accordance to the above mentioned biological levels like mRNA and proteins, the sum of all metabolites is referred as the metabolome. Consequently to transcriptomics and proteomics the analysis of the metabolome is referred as metabolomics. To gain insights into the infection relevant metabolome of the host-pathogen relationship between S. aureus and human lung cells several approaches were developed. First the distribution of the recently identified bacillithiol in different S. aureus strains was investigated with regard to its role during the infection. For that matter a HPLC-methodology was used with fluorescence based detection of labelled low molecular weight thiols (article I: Distribution and infection-related functions of bacillithiol in Staphylococcus aureus). After that the next aim was to reveal the effect of S. aureus on the host cell metabolism. To reduce the complexity of effects on the host cells an artificial model was chosen in a first approach. The lung cells were treated with the staphylococcal virulence factor alpha-hemolysin, a pore forming toxin and a holistic metabolomics approach was performed (article II: Staphylococcus aureus Alpha-Toxin Mediates General and Cell Type-Specific Changes in Metabolite Concentrations of Immortalized Human Airway Epithelial Cells). Using this approach, a protocol for cell culture metabolomics was established and first changes in the host cell metabolome that could be caused by S. aureus were described. However, this only describes specific changes caused by one single virulence factor and does not necessarily describes the reality during a S. aureus infection. Therefore in a next approach, an infection model using a human lung epithelial cell line and the S. aureus strain USA300 was established and used for metabolome analysis. Furthermore a combination of inhibitor treatment and metabolic labelling was used to clarify the metabolic activity in the host cell after exposure to S. aureus (article III: Metabolic features of a human airway epithelial cell line infected with Staphylococcus aureus revealed by a metabolomics approach). Finally this thesis deals with the host-pathogen interaction of B. pseudomallei and its host with a focus on the role of the switch in colony morphology in basic metabolism. Various morphotypes of two strains were generated by nutrient limitation and their uptake of nutrients was monitored. Furthermore the morphotypes were used in in vitro and in vivo infections and subsequently isolated out of the cell line and mice respectively. After isolation, the colony morphology was determined and again the nutrient uptake profile was monitored (article IV: Burkholderia pseudomallei morphotypes show a synchronized metabolic pattern after acute infection). The information provided by this thesis adds a new complexity to the knowledge about the host-pathogen interactions of S. aureus and B. pseudomallei and their hosts. It furthermore lays the groundwork for future studies, which will deal with these and other bacterial host-pathogen interactions in order to understand the interdependencies of infection and metabolism.
An interesting subclass of the SLs are Cers, the simplest SLs. Cers are assigned a special role within SLs because of their involvement in many cellular and biophysical processes.In literature Cers are describe to modulate many events in signaling including apoptosis. Besides its role as second messenger and therefore the involvement in many signal cascades, Cers are also known to be essential in physical modifications and structural alternations of membranes. Such regulatory functions on membrane formation are e.g. domain formation with other lipids (i.g. SM and Chol), phase separation with sterols (Chol), vesicular trafficking, fusion, membrane curvature fluidity and thickness and the induction of membrane leakiness. In contrast to phospholipids, Cers can move from one side of the membrane leaflet to the other, due to their strong hydrophobicity. This movement is called flip-flop or as transbilayer movement and is controversially discussed. Consequently, no exact value has been reported about the flip-flop property of Cers, which probably plays an important role during the transmission of an extra cellular signal through the membrane.In order to probe the biophysical properties of ceramides, a synthetic access to 1-thioceramides (1-SHCer) analogues with different N-acyl chain length has been developed in this study. With 1SHCer the flip-flop was investigated on pre-formed liposomes and the data indicated a very rapid flip-flop of Cers with a half time t1/2 <10s in raft- and non-raft like membrane models. Furthermore, the acyl chain length exhibited no measurable impact on the speed of the flip-flop. Utilizing the same probes the importance of hydrogen bond donor and acceptor properties of Cers upon interaction with sphingomyelin in the presence or absence of cholesterol (Chol) has been probed. Performed fluorescent quenching experiments (P.Slotte) proposed the following relative preference in interaction with pSM:pSM:DAGs > pSM:Cer > pSM:Chol > pSM: 1-pCerSH.Most strikingly, the importance of the 1-OH H-bond acceptor functionality to replace Chol around and above the melting temperature of pSM has been demonstrated. Recently, an unusual subclass of SLs, named 1-deoxysphingoids have come to the foreground, as biomarker for metabolic disorders. 1-doxSA is physiologically generated (10-40nM) due to substrate promiscuity of SPT and shown to be elevated in patients with metabolic disorders. In this study an organic synthetic access to fluorescent DSB derivatives was established, featuring a fluorescent moiety at the lipid tail, such as FITC 26. Comprehensive fluorescent studies of 26 revealed an unusual subcellular distribution. Exogenous 1-doxSA analogues, such as FB1 and 1-doxSA-FITC, enter via specific entry points. During the next few hours these lipids accumulate within the cytosol prior to N-acylation by CerS. Upon N-acylation, the newly formed 1-doxdhCer and its analogues insert into the ER membrane.The fluorescent probe and most likely FB1 analogues accumulate within the late endosomal and lysosomal system, probably via a direct connection with the ER. Analysis of the lipid metabolism of unlabeled 1-doxSA and FB1 revealed a strikingly similar behavior, pointing towards a common pharmacological effect. Complete consumption of TG within 24h in epithelia cells combined with GO analysis of 1-doxSA interacting lipids indicates significant modulation of fatty acid degradation, pointing towards regulation of the energy metabolism. This is in good agreement with the observed induction of autophagy. Together, this rapid and similar metabolic change of both 1-doxSA and FB1, points toward direct 1-doxSA head-group related lipid-protein interaction and less toward the influence of FB1 on CerS activity. This work suggests the biological significance of 1-doxSA as a primary nutrient sensor to maintain nutrient homeostasis and its role in the pathophysiology of metabolic diseases.
This thesis is about the establishment and the application of novel methods and tools that are re-lated to the most widely used enzyme class: hydrolases. It covers all fields from the identification to the application of these valuable enzymes with particular focus on lactonases, acylases and proteases. The activity assay introduced in Article I substantially extends the method toolbox for studies on lactonases and acylases that interfere with the bacterial cell-cell communication system. Article II describes a fully automatized robotic platform that represents the next-level tool for the high-throughput enzyme screening in the microtiter plate format. It was used, for instance, for the screening for improved porcine aminoacylase I variants. Diverse aspects of the protease-mediated hydrolysis of non-resistant proteins for the purification of resistant target proteins are highlighted in Article III.
Enzymatic evolution and the corresponding relationship to substrate scope and catalytic promiscuity were targeted in this thesis. As enzyme examples, pig liver esterase (PLE), oleate hydratases and linoleate isomerases, as well as epoxide hydrolases (EH) and haloalkane dehalogenases (HLD) were used. The substrate scope and the enantiopreference of PLE was analyzed by molecular modeling and substrate docking, since different enantiomeric excesses were detected for the conversion of malonate diethyl esters, depending on the PLE isoenzyme. Additionally, fatty acid converting enzymes with high identity were found and analyzed to comprehend the switch of both activities. Furthermore, the evolutionary connection between EH and HLD was investigated by interconversion studies to implement an HLD acitivity in an EH. By directed evolution and rational design, both possibilities of protein engineering were realized. Finally, a new methodology for targeted, continuous in vivo evolution was established by a temperature-dependent mutagenesis frequency.
Cascade reactions are not only of interest to chemists and biotechnologists, but also to life in general, because every metabolic reaction resembles a cascade reaction. This principle of substrate/intermediate channeling was only adapted by scientists. That way especially one-pot reactions became very attractive as for this no isolation of intermediates is necessary. Furthermore, unstable or toxic intermediates are only produced in low amounts and directly transformed in situ. In this PhD thesis two previously established cascade reactions were subject of further optimization. In the first part, a cascade reaction established in a DFG-funded project (Bo1862/6-1)in cooperation with the Vienna Technical University (Austria) for the production of chiral lactones was further optimized and extended. Therefore, on the one hand the genes encoding the needed enzymes were cloned for co-expression into a single plasmid in different arrangements to be expressed in pseudo-operon mode, with the aim to lower the metabolic burden of the cascade host cell. One out of the welve created constructs showed a reasonable activity of 15.3 ± 1.2 U · gCDW-1. On the other hand, this cascade reaction was aimed to be extended by the use of a hydroxylating enzyme to enable the use of limonene as renewable and chiral precursor for the proposed production of chiral polymers. Therefore, the feasibility of cytochrome P450-monooxygenases was studied. These turned out to be not applicable due to their bad regioselectivity for the hydroxylation of limonene or due to the difficulties of activity reconstitution. As alternative system for an initial hydroxylation step the use of a Rhodococcus equi strain, which was isolated from Cellulosimicrobium cellulans EB-8-4 and which is capable of very regioselective limonene-hydroxylation, was investigated. Therefore, the dioxygenase cluster responsible for the desired reaction was identified and especially the recombinant expression in a suitable host (Pseudomonas putida S12) was further studied. The results from these experiments revealed that the recombinant expression needs to be further optimized to enable the use of the recombinant dioxygenase in combination with the other enzymes for cascade reactions. The third part of this PhD thesis dealt with the immobilization of an established cascade reaction for the synthesis of poly-[caprolactone] precursors. Therefore, the use of a rotating bed reactor (RBR) was investigated. Preliminary studies using single enzymes involved in the desired cascade reaction demonstrated the general feasibility of this reactor concept. Especially the reusability of the catalysts was highly improved, because the catalytic particles were protected very effectively from mechanical forces within the voids of the reactor. For further work-flow optimization the immobilization was transformed into an in situ process by the application of a gas-shear device, which leads to decreased capsule size and thereby to increased mass transfer inside the particles. The developed methods were applied for encapsulation of the cells containing the enzymes needed for the reaction. After additional improvement of the reaction parameters a conversion of 93% (based on substrate depletion) was reached using catalysts produced by the established encapsulation procedure. In summary, the described cascade reactions were successfully optimized by either co-expression, extension applying a dioxygenase or immobilization. Furthermore, the general feasibility of an RBR was demonstrated.
Psychiatric disorders are highly heritable. But the underlying molecular mechanisms are largely unknown or not understood. For many disorders, candidate genes have been proposed which are biologically driven or based on large GWAS studies. In this work different approaches were shown to investigate the impact of genetic risk factors for major psychiatric disorders in the general population. These genetic risk variants include single nucleotide polymorphisms associated with schizophrenia or major depression and were analyzed using the whole-genome information in polygenic scores or candidate marker analysis in GxE studies. Genetic data from SHIP-0 and SHIP-TREND have been used to calculate a polygenic risk score for schizophrenia. Here, the association between this genetic score and brain alterations is shown in three independent samples (SHIP-2, SHIP-TREND and BIG) which revealed no hint of a common genetic basis for schizophrenia and brain structure. These results are in line with other studies that also failed to find a genetic overlap. The same polygenic scores had been used in a PHEWAS analysis in SHIP-0 where an inverse association to migraine was found. This association could be attributed to the NMDA receptor activation via D-serine at the glutamatergic synapse. To assess the impact of environmental factors on the path from genes to phenotype, gene-environment interactions were applied. A significant interaction could be observed between rs7305115 (TPH2) and rs25531 (5-HTTLPR) and childhood abuse on current depression score in SHIP-LEGEND and SHIP-TREND. In summary, genetic variants associated with major psychiatric disorders can exhibit pleiotropic effects on common phenotypes in the general population.
Dilated cardiomyopathy (DCM) is a myocardial disorder characterised by ventricular dilation with reduced left ventricular ejection fraction (LVEF). Immunoadsorption (IA) followed by immunoglobulin (IgG) substitution (IA/IgG) has been shown to be a promising therapeutic intervention to recover myocardial functions in DCM patients. The beneficial effects of IA/IgG therapy are associated with increased LVEF, decreased left ventricular inner diameter at diastole (LVIDd) and reduced myocardial inflammation. Despite knowing the cardiac benefits of IA/IgG, the precise molecular mechanism induced by therapy is still elusive. Additionally, only ≈60 % DCM patients treated with IA/IgG demonstrated improved heart function. Moreover, the reasons for this differential outcome among DCM patients after treatment have not been clearly understood. In this study, efforts were made to uncover the therapy induced proteomic changes in the heart of responders (relative change in LVEF ≤ 20%, LVEF < 5% absolute value) and non-responders using a global proteomic approach. Apart from it, proteomic profiling of endomyocardial biopsies and plasma was performed to find protein biomarker candidates which might be useful to distinguish responder and non-responder DCM patients before immunoadsorption therapy and support a selective and individualized treatment. To reveal therapy induced myocardial proteomic changes, endomyocardial biopsies of DCM patients before and after therapy were compared. LVEF increased (32 ± 8 to 45±7, p<0.002) and LVIDd decreased (66 ± 6 to 60±6, p<0.040) after therapy in responders, whereas non-responders did not show any significant changes in these clinical parameters. To address the changes in the myocardial proteome induced by therapy, a label-free proteomic approach was applied. The most prominent proteomic differences between both subgroups were observed in cytoskeletal, fibrosis, and extracellular matrix proteins. Therapy linked benefit in responders seems to be highly associated with the lower abundance of fibrotic and extracellular matrix proteins which seems to reflect a lower activity of transforming growth factor-β signaling. To elucidate proteomic differences between responders and non-responders at baseline, endomyocardial biopsies and plasma proteome profiling were performed. Responder and non-responder DCM patients did not show any significant differences in the clinical parameters (LVEF, LVIDd, age, inflammation, etc.) before IA/IgG therapy except for disease duration that was in tendency higher among non-responders. Proteomics profiling of endomyocardial biopsies revealed 54 differentially abundant proteins between responders and non-responders. Among those proteins, Protein S100-A8 and kininogen-1 was found higher whereas perilipin-4 was found lower abundant in responders. Plasma profiling of these subgroups revealed five proteins (S100-A8, S100-A9, C-Reactive protein, lipopolysaccharide-binding protein, and cysteine-rich secretory protein) displaying strong discriminative power between responders and non-responders. Higher abundance of Protein S100-A8 was observed in myocardium as well as in plasma among responders. Protein S100-A8 might be a potential candidate to distinguish responders and non-responders at baseline, and its potential utility at clinical levels must be evaluated. The last objective of the thesis was to establish a workflow for the relative quantitation of phosphopeptides for samples generally obtained in small amounts like myocardial biopsies. To address this question, optimization was performed with HL-1 cardiomyocytes using a PolyMAC phosphopeptide enrichment kit and the effect of TGF-β1 on the phosphoproteome was evaluated as a proof-of-principle study. Using only 200µg protein of each sample up to 2000 phosphopeptides with an efficiency of >90 percent could be covered. In total, upon TGF-β1 incubation alterations of 214, 92, and 53 phosphopeptides were observed after 1, 6 and 24 hours, respectively. Differentially altered phosphopeptides belonged to many signaling pathways including the ubiquitin-proteasome pathway, cytoskeletal regulation by Rho GTPase, calcium signaling, and TGF-β signaling. Thus, in this study a workflow for relative quantitation of phosphopeptides was established that may be later applied to precious biopsy samples. Along with this, TGF- β1 induced phosphoproteome was analysed in HL-1 cardiomyocytes.
Heart Failure is currently the most common cardiac disorder and a major public health concern worldwide. The adult mammalian heart harbors a subpopulation of cardiac progenitor cells (CPC) that are capable of improving cardiac function. The scope of this study was to delineate the molecular phenotype of a subpopulation of CPCs characterized by the expression of the stem cells antigen-1 surface marker (Sca-1+) and to further identify molecular alterations occurring under heart failure conditions. In order to understand the underlying cellular mechanisms an integrated approach of proteomics and transcriptomics-based techniques were employed. The first step towards achieving this goal was to unravel the native Sca-1+ cell characteristics of freshly isolated progenitor cells derived from healthy adult murine hearts. The proteome map of Sca-1 cells was established using a gel-based mass-spectrometry (gel LC-MS/MS) approach. For better interpretation, a comparison with the protein profiles of cardiomyocytes and Sca-1- cells obtained under similar experimental conditions was performed. All three cell-types were morphologically different in size and structure, which was also evident from their protein expression profiles. We observed that Sca-1+ cells lack endothelial-like and cardiac contractile phenotypes, unlike Sca-1- cells and cardiomyocytes, respectively. Functional assessment of both protein and gene expression profiles revealed a possible role of Sca-1+ cells in cell adhesion, migration, and proliferation. CPC remain in a dormant state under physiological condition unless challenged by myocardial injury. Previous studies revealed that resident Sca-1+ cells home to the injured myocardium but not to the healthy heart and further differentiate into functional cardiomyocytes. We investigated the molecular background of this behavior of adult Sca-1+ cells under heart failure condition which might provide a better insight into their cardiogenic potential in a pathological milieu. The double transgenic α-myosin heavy chain (MHC)-cyclin T1/Gαq overexpressing mouse was chosen as a model for heart failure. Using the comparative gene expression profiling we could detect the differential regulation of 197 genes with at least a 2-fold difference. Among these BDNF mRNA levels were 5-fold higher in the Sca-1+ cells derived from transgenic mice (Cyc+) in comparison to that of wild-type controls (Wt+). This difference was also observed at protein level. The substantially higher expression of BDNF during heart failure prompted us to investigate its regulatory effect on Sca1+ cells. In this current study we were able to show that small amounts of exogenous BDNF stimulated the migratory potential of Cyc+ cells. This effect was not seen in treated Wt+ cells. Furthermore, pulsed SILAC was employed to monitor BDNF mediated changes following treatment. After BDNF treatment, 58 proteins were differentially regulated of which proteins related to cell proliferation were reduced in level in Cyc+ cells while they displayed increased levels in Wt+ cells. Findings from bromodeoxyuridine (BrdU) assays and immunoblotting indicated that BDNF might initiate a differentiation program by repressing cell proliferation in Cyc+ cells. Taken together, it could be shown that the BDNF effect on protein synthesis of Cyc+ and Wt+ cells varied considerably, suggesting an improvement of the cardiogenic potential of Sca-1+ cells under pathological conditions. Aldosterone levels are known to be elevated during heart failure. In this part of study it was hypothesized that endocrine factors associated with heart failure might influence the migration of CPC, thereby possibly restoring the cardiac function of diseased hearts. It could be shown that high concentrations of aldosterone, similar to those found in the plasma of heart failure patients, induced the migration of Sca-1+ cells by up to 60% when compared to control, while physiological levels had no significant influence. In addition, it could be demonstrated that the aldosterone stimulus led to the activation of the mineralocorticoid receptor (MR) expressed on Sca1+ cells, which in turn facilitated migration. This was supported by application of MR antagonist eplerenone, which significantly reduced the aldosterone-induced increase in cell migration while a glucocorticoid antagonist exhibited no inhibitory effect. Hence, the results support the potential role of aldosterone in the mobilization of CPC. It is currently believed that the beneficial effects of cell-based therapies on cardiac repair are imparted to a large degree via paracrine mechanisms. We therefore focused on understanding the influence of pathophysiological levels of aldosterone on the extracellular environment of Sca-1+ cells. MS-based secretome profiling of cells treated for 24h with aldosterone treatment revealed higher levels of proteins associated with extracellular matrix remodeling and IGF signaling. Additionally, galectin-1 and gelsolin were significantly increased in level under pathological conditions indicating a possible paracrine tissue repair of Sca-1+ cells. To conclude, the global proteome and transcriptome profiles generated here revealed the molecular phenotype of Sca-1+ cells which may be used for future reference. The comparative microarray study provided deeper insight into the endogenous changes in mRNA expression during heart failure and delineated the cardiogenic characteristics of Sca-1+ cells. Moreover, the data presented here shed new light on the potential role of BDNF in regulating the mobilization and proliferation of CPCs. Our study on the influence of aldosterone on the migration and the extracellular proteome of CPCs provided new insights on the beneficial effects of this mineralocorticoid on cardiac cells.
Ischemic stroke is the second leading cause of death worldwide and a disease with a variety of risk factors including hypotension, nutrition/obesity, and smoking but also increased age. In an ageing society stroke is a great challenge and leaves the survivors with disabilities. The aim of this dissertation was to investigate the immunologic changes post ischemic stroke, in order to use a better understanding for new therapeutic approaches as well as for improvement of translation of results from bench to bedside. Ischemic stroke leads to a local and peripheral immune activation. On the other side an immune dysfunction/suppression occurs, that leads to a higher risk of stroke-associated infections. In this dissertation, a long-lasting elevation of HMGB1 after stroke and a correlation with blood leukocyte numbers could be shown. HMGB1 seems to be an important mediator of an endogenous inflammation and an interesting target for post-stroke immunomodulation. In a further study we showed that the quality of the immune response of infiltrating T cells has an impact on the neurologic outcome and functional recovery after experimental stroke. Importantly, a mechanism of how infections, mimicked by LPS injections, could worsen the outcome of stroke patients was revealed. In the context of stroke-induced immunosuppression regulatory T cells as an immunosuppressive T cells subset seem to not play a role as their suppressive capacity is reduced after stroke. Interestingly, the CD39 expression on Tregs is similarly increasing with age in humans and mice. This shows the importance of an age equivalent in experimental studies. In search of predictors for the outcome after stroke as well as the risk of infections, we performed single nucleotide polymorphism genotyping in the IL-1RN and TLR4 gene of stroke patients. Functional significant variants in the IL-1RN and TLR4 genes may have an impact on outcome and systemic markers of inflammation post stroke but these findings need to be replicated in studies with much larger cohorts.
The thesis deals with ions stored in an electrostatic ion beam trap. In the first part of the thesis the so-called self-synchronization effect is discussed. It is demonstrated that the time a bunch of injected ions is conserved by the self-synchronization effect depends on the number of injected ions. In the second part of the thesis the cooling of small anionic cobalt and copper clusters is addressed. Measurements on anionic copper clusters consisting of four to seven atoms are presented and the decay of hot clusters is observed in order to draw conclusions on the internal temperature and the cooling process itself. Afterwards measurements on Co4- are discussed and a measurement scheme based on laser induced delayed electron emission is presented enabling to monitor the internal energy distribution of the clusters over storage time in a temperature-controlled environment. The cooling of initially hot clusters as well as the heating of initially cold clusters were observed.
Staphylococcus aureus is present in around a third of the human population as a constant commensal in the anterior nares, in a third as an intermittent commensal, and a third are non-carriers. However, S. aureus is also a dangerous pathogen, responsible for many types of infections. Recently, the emerging of methicillin-resistant S. aureus strains has aggravated the health problem. Treating infections caused by the invasive strains has become ineffective with conventional antibiotics. Noticeably, transmission of S. aureus has occurred not only in healthcare settings but also in the community; furthermore, transmission between humans and domestic animals has been reported. Although studies about host-pathogen interactions of S. aureus have advanced our knowledge in the last decades, we still have not fully understood mechanisms of the immune system in responses to S. aureus. The aim of this study is to unravel interactions of the human adaptive immune system to selected S. aureus virulence factors. In particular, the study focuses on two aspects: the reaction of human antibodies to the bacterial extracellular proteins in S. aureus-induced furunculosis with an emphasis on Panton-Valentine Leukocidin and responses of the adaptive immune system to membrane-bound lipoproteins of S. aureus. Furunculosis is a variety of hair follicle infection in which S. aureus is one of the chief causal pathogens involved. The corresponding bacterial strains are generally capable of producing of a pore-forming toxin, known as Panton-Valentine Leukocidin (PVL). Recently, the emerging of pvl-positive methicillin-resistant S. aureus has become a problem for treating the bacterially caused furuncles. Colonization with the bacteria is a risk factor for development of chronic or recurrent boils. It is not yet known why furunculosis patients are largely infants or young adults. In this context, we untangled the responses of antibody IgG antibodies to S. aureus extra-cellular factors, notably the PVL toxin, in families in which the patients were children. Multiplex PCR demonstrated that S. aureus clones, isolated from the patients’ wounds but also from the nares of family members, harbored genes coding for PVL toxin. Spa-typing highlighted that bacterial genotypes were very similar in each family. This suggests that transmission of pvl-positive S. aureus took place between family members. The finding also raises the question why only the young patients but not family members who were colonized by the same S. aureus clones suffered from furunculosis. 2D immune proteomics procedures showed a tendency of higher IgG titers against bacterial virulence factors in family healthy members than in patients. PVL-specific antibodies were measured using ELISA, in which patients’ PVL-specific IgG titers were low. This supports the idea that antibodies, probably in conjunction with T cells, might contribute to clinical protection in furunculosis. This research will serve as a foundation for future studies, in which our results should be validated in a larger cohort. Among S. aureus’ virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of the complex of innate immune receptors termed Toll-like receptors (TLR) 2 and 6. This study addressed the specific B-cell and T-cell responses to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of serum antibody (IgG) binding to the S. aureus lipoproteins were very low or even unmeasurable in healthy individuals except for the lipoprotein SaeP. Only patients with cystic fibrosis or epidermolysis bullosa who were heavily exposed to the bacteria, generated an antibody response also to lipoproteins. Proliferation assays and cytokine profiling data showed only subtle responses of T cells in healthy individuals; three out of eight tested lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may be inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells. The main findings implicate that family members can serve as S. aureus reservoirs causing recurrent furunculosis in young patients and that antibodies may provide partial protection from such infections by S. aureus. We have found that, different from proteins that are secreted by S. aureus, lipoproteins which anchored in the bacterial cell membrane, do not trigger strong responses from the human adaptive immune system. This suggests that these proteins remain mostly hidden in the bacterial cell-wall.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancy and projected to be the third leading cause of cancer related death by 2030. Despite extensive knowledge and insights into biological properties and genetic aberrations of pancreatic ductal adenocarcinoma cells, therapeutic options remain temporary and ineffective. One plausible explanation for the futile response to therapy is an insufficient and nonspecific delivery of anticancer drugs to the tumor site. Superparamagnetic iron oxide nanoparticles (SPION) coupled with siRNA targeted against the cell cycle specific serine-threonine-kinase, Polo-like kinase-1 (siPLK1-StAv-SPIONs) could serve a dual purpose for delivery of siPLK1 to tumor and noninvasive assessment of delivery in vivo. siPLK1-StAv-SPIONs were designed as theranostics to function via a membrane translocation peptide (MPAP-) as well as a tumor selective peptide (EPPT-1) to increase intracellular delivery and tumor specificity, respectively. In vitro and in vivo experiments using a syngenic orthotopic PDAC model as well as the endogenous LSL-KrasG12D,LSL-Trp53R172H,Pdx-1-Cre model revealed significant accumulation of siPLK1-StAv-SPIONs in PDAC resulting in efficient PLK1 silencing. Tumor specific silencing of PLK1 halts tumor growth, marked by decrease in tumor cell proliferation and increase in apoptosis. siPLK1-StAv-SPIONs are well tolerated with no observed systemic side effects. Our data suggests, siPLK1-StAv-SPIONs with dual specificity residues for tumor targeting and membrane translocation, represent an exciting opportunity for targeted therapy in PDAC.