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Next Generation Sequencing (NGS)-technologies developed very fast in recent years and is used widely in current research areas. The aim of this study was to use NGS (i) for the identification of pathogens in outbreaks and (ii) for the identification of virulence-relevant sequencepolymorphisms when comparing whole genome sequences. Therefore, a previous developed workflow was used to identify a new virus of the family Bornaviridae. The generation of whole genome sequences elucidated the molecular epidemiological connection of infection of variegated squirrels (Sciurus variegatoides) and three human cases of fatal encephalitis. By generating the whole genome sequence of a Porcine Epidemic Diarrhea Virus (PEDV) in Germany it was possible to find difference compared to circulating high virulent strains in the USA. This led to potential virulence marker to distinguish strain in the USA and Germany. Connections between sequence variation and virulence were further investigated for the bovine viral diarrhea virus 2c (BVDV-2c), cowpox viruses (CPXV) and classical swine fever virus (CSFV). Here, for a highly virulent BVDV-2c strain a mixture of different genome structure variants could be found. The majority of these genomes harbors a duplication within the p7/NS2 coding region and might cause a high virulence. For CPXV virus isolated of different hosts were analyzed and a correlation between genome sequence and the A-type inclusion body phenotype could be found. Furthermore, several deletion/insertion events were detected which might influence the virulence of these strains. Finally, the virus population of CSFV strains in pigs was characterized. However, the population of the inoculum as well as of acute-lethal and chronically infected animals gave no indication that the virus itself causes the different types of disease outcome. In conclusion, this thesis shows the great potential of NGS for virus identification and characterization. Furthermore, it makes the identification of potential virulence marker possible which subsequently can be analyzed by reverse genetics.
The present work examines the decision-making process of clinicians and managers in terms of pricing. This dissertation attempts to make often unconscious processes in both the clinical and economic areas more transparent. Due to this increase in transparency and an illustration of the overall decision-making process, a prioritization of the decision-drivers is enabled. Similarily, overall decision-making on stakeholder level is improved. The findings of this work are based on a dual sample: The paper primarily combines qualitative expert discussions in the clinical field with a quantitative manager survey. Therefore, the advancement of knowledge is specifically furthered in terms of pricing decision-making, as well as specific economic and thematic aspects. These form the basis for clinical/managerial decision-making. Since such specific understanding enables better acting and reacting during the negotiation processs, clients of medical device companies (eg clinics) could additionally benefit from improved understanding. Although the focus of this work rests on the clinical area, this paper also examines how companies of the medical technology industry could offer more economic medical products by means of using the instrument of pricing. Based on an interdisciplinary approach and a collection of primary data, this work also discusses a solution approach for corporate/entrepreneurial inefficiencies discovered in this paper. A final conceptual model illustrates different value allocations from both stakeholder groups (clinicians vs. managers). With regard to the research focus, the conceptual model is understood as a holistic solution for recognizing and correcting business gaps in the areas of knowledge transfer, innovation, knowledge of markets and standardization of processes.
Today the process of improving technology and software allows to create, save and explore massive data sets in little time. "Big Data" are everywhere such as in social networks, meteorology, customers’ behaviour – and in biology. The Omics research field, standing for the organism-wide data exploration and analysis, is an example of biological research that has to deal with "Big Data" challenges. Possible challenges are for instance effcient storage and cataloguing of the data sets and finally the qualitative analysis and exploration of the information. In the last decade largescale genome-wide association studies and high-throughput techniques became more effcient, more profitable and less expensive. As a consequence of this rapid development, it is easier to gather massive amounts of genomic and proteomic data. However, these data need to get evaluated, analysed and explored. Typical questions that arise in this context include: which genes are active under sever al physical states, which proteins and metabolites are available, which organisms or cell types are similar or different in their enzymes’or genes’ behaviour. For this reason and because a scientist of any "Big Data" research field wants to see the data, there is an increasing need of clear, intuitively understandable and recognizable visualization to explore the data and confirm thesis. One way to get an overview of the data sets is to cluster it. Taxonomic trees and functional classification schemes are hierarchical structures used by biologists to organize the available biological knowledge in a systematic and computer readable way (such as KEGG, GO and FUNCAT). For example, proteins and genes could be clustered according to their function in an organism. These hierarchies tend to be rather complex, and many comprise thousands of biological entities. One approach for a space-filling visualization of these hierarchical structured data sets is a treemap. Existing algorithms for producing treemaps struggle with large data sets and have several other problems. This thesis addresses some of these problems and is structured as follows. After a short review of the basic concepts from graph theory some commonly used types of treemaps and a classification of treemaps according to information visualization aspects is presented in the first chapter of this thesis. The second chapter of this thesis provides several methods to improve treemap constructions. In certain applications the researcher wants to know, how the entities in a hierarchical structure are related to each other (such as enzymes in a metabolic pathway). Therefore in the 3 third chapter of this thesis, the focus is on the construction of a suitable layout overlaying an existing treemap. This gives rise to optimization problems on geometric graphs. In addition, from a practical point of view, options for enhancing the display of the computed layout are explored to help the user perform typical tasks in this context more effciently. One important aspect of the problems on geometric graphs considered in the third chapter of the thesis is that crossings of edges in a network structure are to be minimized while certain other properties such as connectedness are maintained. Motivated by this, in the fourth chapter of this thesis, related combinatorial and computational problems are explored from a more theoretical point of view. In particular some light is shed on properties of crossing-free spanning trees in geometric graphs.
A method employing labeling of cell-surface proteins with Sulfo-NHS-SS-biotin and subsequent affinity enrichment with NeutrAvidin has been optimized in order to make cell-surface proteins from Gram-positive bacteria reliably accessible to quantitative mass spectrometric analyses. The optimized biotinylation approach was applied for analysis of the lipoproteome from S. aureus and S. pneumoniae on a global scale and the influence of mutations in the lipoprotein maturation pathway on the cell-surface and exoproteomes of both species was investigated. The biotinylation approach was integrated into a proteomic workflow that employs metabolic labeling with heavy nitrogen for relative protein quantification to investigate proteomic differences between S. aureus in a biofilm model and its free-floating, planktonic counterparts.
Although the Pleistocene deposits exposed in the steep coastal cliffs of Mecklenburg-Vorpommern have been studied for more than a century, the depositional conditions of many lithostratigraphic units remain unclear. There is, in particular, a question whether the individual tills (locally more than 9 successive till units) are mainly subglacial deposits or resedimented (mass flows) in origin (at least in part). The Pleistocene deposits preserve information concerning the former glacial depositional processes. Detailed micromorphological analysis of these deposits can provide key information regarding these processes and thereby aid in the reconstruction of former glacial environments. The island of Rügen is located on the southwestern Baltic Sea coast and was situated in the marginal zone of the Scandinavian Ice Sheet during the last glacial period (Weichselian). Therefore, the region is considered as an ideal area for reconstructing the complex fluctuations in the position of the margin of this ice sheet as it expanded across the Baltic Sea and into northern Germany. Successive glacial advances and retreats of the ice sheet can be reconstructed by specific glacial sedimentation processes and flow-direction criteria derived from a variety of glacial deposits. The investigation area is located near Sassnitz on Rügen, where an imbricated and folded Weichselian succession disconformably overlies Maastrichtian chalk bedrock. The individual till units were sampled for micromorphological analyses to identify the former depositional conditions. Detailed description of the sedimentology and variation in facies, the description of macroscale deformation structures provides the context for the detailed micromorphology study. The three dimensional analysis of the microfabrics is based on the microstructural mapping methodology which enables the identification and interpretation of polyphase deformation within subglacial sediments.
Telemedicine at the Emergency Site – Evaluated by emergency team members in simulated scenarios
(2015)
The hypothesis of this study states that emergency medicine can benefit from telemedicine, whenever paramedics at a remote emergency site request consultation or mentoring by a distant emergency doctor. The hypothesis was semi-qualitatively evaluated in accordance with the protocol of the EU project in the setting of a medical simulation centre. Paramedics encountered simulated standardized emergency case scenarios, connected for teleconsultation and telementoring with emergency doctors by video and audio link through a newly developed real-time HD-video system called LiveCity camera. Paramedics and emergency doctors regarded the simulated scenarios as realistic and relevant and took the simulation seriously. Thus,the following conclusions can be drawn: 1.) Emergency team members encounter situations at the emergency site, in which they would like to get help by a more experienced colleague, especially help with diagnostics and treatment. 2.) The telemedical contact to an emergency doctor makes paramedics feel confirmed in their work, more secure, even in legal aspects. Paramedics do not feel controlled by telemedicine or like a puppet on a string. Their relationship to the patient is not mainly deranged or interfered by the doctor and their course of action is not mainly disrupted. The tele-emergency doctors do not feel like puppet masters and continue feeling as doctors and do not perceive themselves as interferer within the emergency team. 3.) Emergency team members call for a telemedical system providing transmission of vital signs as well as audio- and video-connection. 4.) The LiveCity camera is an effective telemedical tool. The audio quality is good and the orientation on the screen is easy. Paramedics state, that filming the emergency site is easy, does not restrict the field of vision and paramedics can communicate the emergency doctors everything they want to show and tell. Thus the emergency doctors get additional information. While the LiveCity camera is mostly perceived as not too heavy, the LiveCity camera is not easy to operate, very failure-prone and can derange the communication among team members at the emergency site. Nevertheless, the LiveCity camera is not perceived as an additional burden. 5.) Telemedicine is predominantly and largely appreciated by the members of the emergency team. Connecting the tele-emergency doctor to the remote paramedics leads to a perceived faster start of the therapy and is considered as helpful, improving the situation and the quality of patient care. The adherence to medical guidelines and therefore the quality increased, when the paramedics were connected to an emergency doctor through the telemedicine connection. In general, the quality of diagnostics, the correctness of diagnosis and the quality of therapy were rated higher. The majority of paramedics would call a tele-emergency doctor in cases, they wouldn´t normally activate medical support. The emergency team members largely agree in perceiving the tele-emergency doctor system as useful, and they can imagine, working in a tele-emergency system. As a conclusion, the general hypothesis of this study is mainly and in many items supported: Emergency medicine benefits from telemedical support via video- and audio link as studied here with a newly developed real-time HD-video system called LiveCity camera, whenever paramedics at a remote emergency site request consultation or mentoring by a distant emergency doctor.
Staphylococcus aureus can be a harmless colonizer of the human body, which colonizes about 20-30% of the population. If S. aureus overcomes the outer physical barrier of the body, comprised of the skin and mucous surfaces, it can also cause severe diseases such as endocarditis, pneumonia, or sepsis. S. aureus possesses a variety of secreted and surface bound virulence factors to mediate attachment and invasion into the host, to disseminate an infection and to modulate and evade the immune system. But not only the huge amount of virulence factors turn S. aureus into a dangerous human pathogen, also its resistances to a broad spectrum of commonly used antibiotics make infections hard to treat. During the last years it became apparent that S. aureus can be internalized by as well as replicate and persist in professional and non-professional phagocytic cells. It is suggested that the intracellular compartment protects S. aureus from antibiotic treatment and the immune system. To accomplish the adaptation to the intracellular compartment, S. aureus needs to regulate its gene expression by regulatory systems. One of these regulators is the alternative sigma factor SigB, which directly and indirectly regulates the expression of about 200 genes in vitro. However, the stimuli leading to the activation of SigB in S. aureus are barely known and also its role during an infection varies, depending on the S. aureus strain and infection model used. Therefore, the importance of SigB during the early adaption of S. aureus to the intracellular environment should be elucidated using a cell culture infection model. First, the existing cell culture infection workflow had to be modified to improve the data analysis and to increase the yield of identified proteins to comparatively monitor the adaption reaction of S. aureus HG001 and its isogenic ΔsigB mutant to the intracellular milieu of S9 human bronchial epithelial cells. The proteome analysis in conjunction with RT-qPCR analysis of the wild type and the ΔsigB mutant revealed a fast and transient activation of SigB directly after internalization. Quantitative analysis of the intracellular bacterial titer demonstrated a requirement of SigB for intracellular replication. Differences in the proteome composition of the ΔsigB mutant in comparison to the wild type after internalization reflected the different growth rates, resistance to antibiotics and toxic compounds, adaptation to oxidative stress, and protein quality control mechanisms. The accessory gene regulator (Agr) is like SigB also a global regulator of gene expression in S. aureus. To elucidate possible benefits in the intracellular survival of the co-occurrence of S. aureus wild type and Δagr mutant cells, like it can be found in sites of an infection, a co-infection assay was established. With the co-infection assay the simultaneous and competitive intracellular survival in comparison to the individual intracellular survival was followed for three days post-infection (p.i.). The single and the co-infection revealed that the wild type was able to replicate more efficiently during the first hours p.i. than the Δagr mutant, but the mutant was able to survive more efficiently. The extracellular proteome of S. aureus represents the key compartment for virulence factors. Virulence factors are secreted or bound to the surface of the S. aureus cell. With the infection workflow applied in this study, secreted proteins are lost during the enrichment of the intracellular bacteria for proteome analysis. Therefore, no information about the levels or the regulation of virulence factor expression can be acquired in the cell culture infection model using cell sorting approaches. Hence, the extracellular proteome of S. aureus was analyzed in vitro from shake flask experiments. To get a comprehensive overview of the regulatory impact of different global regulators onto the secretome, S. aureus LS1 mutants lacking the global regulators Agr, SarA and SigB were compared to the respective wild type. Additionally the protein level of the secretome of the well characterized and frequently used S. aureus strains 6850, CowanI, HG001, LS1, SH1000, and USA300 was comparatively analyzed. This project was performed in collaboration with the group of Prof. Löffler from the Institute of Medical Microbiology in Jena. The data of the extracellular proteome generated in this thesis were combined with phenotypic and toxicity data to explain strain differences in invasiveness, cytotoxicity, phagosomal escape, and intracellular persistence in infection experiments.
Microbial infections can be either caused by a single species or complex multi-species consortia. One of the most prominent opportunistic human pathogens leading to mono- or mixed-species infections is the Gram-negative bacterium Pseudomonas aeruginosa. Understanding the molecular basis of its adaptation to infection-related stresses is an essential prerequisite for the prevention and treatment of P. aeruginosa infections. We therefore employed state-of-the-art proteomics approaches to elucidate the molecular adaptation mechanisms of P. aeruginosa to infection-related conditions. Moreover, structure, function and interaction of complex microbial consortia containing P. aeruginosa and causing catheter-associated urinary tract infections were investigated by metaproteomics analyses. Our investigations revealed that the adaptation of P. aeruginosa during infection is either based on gene expression changes caused by environmental signal integration or by gene mutations leading to a selective advantage in a particular host environment. In study I, investigating the proteome response of P. aeruginosa biofilms to the clinical relevant antibiotic ciprofloxacin, global changes in the protein profile were observed. Ciprofloxacin induced the expression of proteins involved in the Lex-induced SOS-response, drug efflux pumps and gene products of the ciprofloxacin-responsive prophage cluster and repressed the expression of porins and DNA-binding proteins. In study II the transcriptome and proteome of two clonal P. aeruginosa lineages during long-term colonization of cystic fibrosis (CF) patient’s lungs were analyzed. Point mutations in global regulator genes, i.e. retS, gacS, and gacA, were identified by genomic sequencing. Inactivation of RetS, found two years after the initial colonization, induced the expression of genes involved in chronic infections and coding for the type 6-secretion system (T6SS). Additional mutations in the GacS/GacA two-component regulatory system (TCS) were found to repress the expression of T6SS proteins and to induce the expression of proteins belonging to the type 3-secretion system (T3SS). In study III we elucidated the niche-specific adaptation of P. aeruginosa isolates from different infection sites by investigating their protein expression patterns and glucose metabolic fluxes. We could show that isolates from the urinary tract express a higher amount of proteins involved in the acquisition of micronutrients (i.e. iron) and carbohydrates compared to isolates from the CF lung. In study IV 16S rDNA sequencing and metaproteomics were employed to demonstrate that the investigated CAUTI-related biofilms consisted of two to five different species with one or two species dominating the mixed community. Following this line of research, we investigated in study V structure and function of a biofilm of a long-term catheterized patient, which was predominantly composed of P. aeruginosa and Morganella morganii, but also contained a minor proportion of the obligate anaerobe Bacteroides sp.. The comparison of in vivo and in vitro protein expression profiles of P. aeruginosa and M. morganii indicated that iron and carbohydrates are the major growth-limiting factors in the bladder. These results indicate different nutritional strategies of the two pathogens in the bladder environment. A comparison of urinary protein profiles of healthy persons and catheterized patients suggested that the human innate immune system is induced by CAUTIs. Moreover, numerous proteins involved in nutritional immunity, e.g. iron-, calcium- and magnesium-binding proteins, were found to be more abundant in the urine of catheterized patients. A follow-up (meta)proteomics study (study VI) aiming at the elucidation of interspecies interactions during multi-species infections indicated that the urease-positive uropathogen Proteus mirabilis induces the precipitation of metal ions by urine alkalization and thereby limits the availability of these important micronutrients for other co-infecting bacteria. This limitation seems to be sensed by the P. aeruginosa PhoP-PhoQ two-component system (TCS) leading to an increased resistance to antimicrobial peptides and biofilm-forming capacity of the pathogen. Also during co-cultivation of P. aeruginosa with Staphylococcus aureus a slight increase in the expression of the PhoP-PhoQ TCS and the alkaline protease could be observed (study VII). In study VIII a combined metagenomics and metaproteomics approach was employed to investigate structure and function of the lichen Lobaria pulmonaria, a complex consortium consisting of a fungus, an algal partner, cyanobacteria, and a highly diverse bacterial microbiome. The results presented in this work contribute to a better understanding of the manifold and complex bacterial adaptation mechanisms to infection-related and environmental stress and thereby foster the development of novel treatment and prevention strategies.
With the growing importance of advanced lighting technologies, customers expect additional functionality and higher comfort from fluorescent lamps. However, the ability to regulate light intensity (dimmed operation), in particular, exerts enormous stress on fluorescent lamps’ electrodes, leading to increased electrode erosion and significantly reduced lifetimes. During the operation of a fluorescent lamp, free barium (the main compound of the electrode emitter) is produced at the electrode responsible for lowering the work function in order to enable energy-efficient and durable electrodes with lifetimes of up to 20,000 hours. Despite their relatively long lifetimes, electrodes remain the lifetime-limiting factor of a fluorescent lamp. Therefore, for practical applications (e.g., maintaining quality control, adjusting operational parameters, and evaluating new electrode designs), electrode erosion is of special interest. The actual erosion-measurement methods determine a time-averaged erosion level over several hundred operation hours. Thus, a quasi-instantaneous measuring method (short measurement) is still necessary to determine erosion during operation. Such a method would allow us to compare erosion under different discharge conditions (currents, frequencies, or heating currents) from the same electrode in the same lamp. This work focuses on the determination of absolute electrode erosion during the stationary operation of commonly used fluorescent lamps. Commercial T8 lamps (fluorescent lamps with a diameter of 8/8 inch) are investigated at the operating mode of commonly used electronic ballasts with frequencies of several kHz. Operations under standard and dimmed conditions with an additional heating current to reduce electrode erosion are investigated. Electrode erosion is characterized by the erosion of barium, the main compound of the electrode. Therefore, laser-induced fluorescence (LIF), which is the most sensitive method for this application, is applied to determine the absolute densities of the eroded barium in the electrode region. These densities are affected by the plasma in the electrode region and do not directly represent the absolute barium erosion. To overcome this limitation, a new method based on a special measurement technique in combination with a barium-diffusion-model is developed to determine the absolute barium erosion based on the measured densities. It has been found that the barium densities in the electrode region are lower than the equilibrium pressures produced by the reduction of the barium oxide. This could be caused either by a reduced reaction rate, the reduced diffusion of the reactant (primarily barium oxide) or by reduced barium transport through the porous emitter. However, these results suggest that barium erosion depends on temperature and emitter structure, which vary over an electrode’s lifetime. For currents significantly higher than the nominal lamp current, a drastic increase in emitter evaporation is found. Such, an increase in the lamp current from 300 mA to 500 mA leads to an increase in emitter evaporation by a factor of five. Using the lamp for a long period of time under these conditions therefore reduces the lifetime by a factor of five. Notably, at this dramatically increased erosion level, the hot spot temperature only increases from 1120 K to 1170 K. Investigation of various frequencies from 50 Hz to 5 kHz revealed no significant dependence of emitter evaporation on frequency.
Geopolymers (GPs) are inorganic binders created by adding alkaline solution (e.g. KOH) to silicates such as furnace slag, fly ash or clay to dissolve Si and Al that polymerises and precipitates to form an inorganic binder material while hardening. GP properties are similar to ordinary Portland cement regarding their high compressive strength or low shrinkage but they are particularly notable for a high resistance to acid and fire. However, the most significant advantage of GP cements is their low CO2 footprint. The most common clay used as GP raw material is kaolin. The aim of this study is to investigate the suitability of illitic clays as a cheaper alternative to kaolin and determine the necessary preparation steps required to produce effective GP binder materials. Three clays dominated by dioctahedral 2:1 layer silicates, in particular interstratifications of mica and smectite were investigated: (1) Illitic clay from Friedland, Northern Germany, containing an irregularly stacked illite-smectite interstratification (R0 I-S), (2) rectorite from Arkansas, USA, as a regular interstratification of mica and smectite, and (3) clay stated as “sárospatakite” from Füzérradvány clay deposit, Northern Hungary, containing a long range ordered I-S (R3). The three types of I-S interstratification-rich clays were extensively characterised and the Friedland clay, as the most probable raw material for GP production, was studied in more detail including several size fraction analyses. These results are used to investigate and determine the parameters necessary to produce suitable precursors for GP binders. Different approaches of clay activation to yield a highly reactive material by milling and heating were examined. Milling was found to be suitable as a preparation step after heating breaking up sintering aggregates to create pathways for the alkaline solution, but not as a substitute for heating. Important parameters for the precursor design such as temperature, time, and heating rate are determined and discussed. Geopolymerisation is considered to be a multi-parameter system and is influenced strongly by the degree of dehydroxylation, Si:Al ratio, or amount of 5-fold coordinated Al. However, in contrast to kaolin-based systems, none of these parameters explain why the illitic Friedland clay heated to 875 °C was found to be most suitable for GP binders. Based on leaching experiments and specific surface area (AS) measurements of the heated Friedland clay, a conceptual model is presented to explain the observed relationship between the heating temperature and the subsequent compressive strength of the GP cement. An optimum between the counteracting reactions of decreasing AS (fewer particles must be covered with GP phase) and decreasing Si+Al dissolved (less GP phase created) is necessary, which exists at 875 °C for the Friedland clay. In this state enough GP phase is created to bind all remaining sintering aggregates to form a cement with high compressive strength. This relationship can be expressed as (Si+Al) / AS (sum of dissolved Si and Al divided by the surface area of grains that must be covered with GP phase), and can be used as a predictive tool for determining the optimal heating temperature. The results presented in this thesis indicate that illitic clays are suitable raw materials as GP binders if the necessary preparation steps of dehydroxylation, sintering and grinding are made. Proxies used to evaluate the optimal conditions for making GP binders are determined including the (Si+Al) / AS ratio as a key relationship that controls the cementation process and determines its ultimate hardness.