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Antibiotic uptake and protein quantification in Gram negative bacteria using Mass spectrometry.
No full textMany antibiotics have intracellular targets and their pharmacological action is well
characterized. However, the antibiotic uptake mechanism in bacteria is not fully resolved.
Therefore, a greater efforts are required to understand the uptake of these drugs
across the outer membrane in order to counteract bacterial resistance. A robust and sensitive technique like mass spectrometry can effectively be used to detect drug accumulation in bacterial cell. In a nutshell, the first part of research will be focused on the method to detect antibiotics in bacterial cell and the second part, will be focused on quantitative number of active influx porin in bacterial mutants.
The first part of this thesis provides insight on how mass spectrometry can be used to study intracellular accumulation of antibiotics in Gram-negative bacteria. In this study, a high-throughput method using UHPLC-ESI-QTOF/MS was developed and different
antibiotics are (Ciprofloxacin, Levofloxacin, Tetracycline, Linzolide, Fleroxacin and addition of other efflux inhibitors) used to measure intracellular accumulation in Escherichia coli. The different mutants of W3110 E. coli, wild-type, acrAB efflux pump KO,ompFompC double porin KO, and ompFompCacrAB triple mutants were used to study differential drug uptake in bacteria. Where the efflux mutant showed two-fold higher antibiotic accumulation when compared to wild-type and double porin mutant. There is an alternative uptake pathway expressed in double porin mutant due to which antibiotic accumulation is observed. Thus, this methodology could be implemented in study of xenobiotics uptake as a strategy to complement the knowledge of the dietary composition in foodomics (Improvement in food and nutrition through process of technology). The second part explains the importance of porins in E. coli and sheds light on correlation of porin regulation between different mutant strains. The study is well understood by using isotope labeled peptide in quantification
Understanding antibiotic permeation through OccD3 of Pseudomonas aeruginosa
No full textPseudomonas aeruginosa utilizes a plethora of substrate specific channels for the uptake of small nutrients. OccD3 (OpdP or PA4501) is an OprD-like arginine uptake channel of P. aeruginosa whose role has been implicated in carbapenem uptake. To understand the mechanism of selective permeation, we reconstituted single OccD3 channels in a planar lipid bilayer and characterized the interaction with Imipenem and Meropenem, analyzing the ion current fluctuation in the presence of substrates. We performed point mutations in the constriction region of OccD3 to understand the binding and translocation of antibiotic in OccD3. By mutating two key residues in the substrate binding sites of OccD3 (located in the internal loop L7 and basic ladder), we emphasize the importance of these residues. We show that carbapenem antibiotics follow a similar path as arginine through the constriction zone and the basic ladder to translocate across OccD3
Trace amine-associated receptor 1 is involved in various aspects of endocrine regulation
No full textTrace amine-associated receptor 1 (Taar1) was identified in 2001 as a receptor related to the serotonin receptor family. Taar1 was found to be expressed in thyroid and pancreas. Therefore, we designed this study in which Taar1 expression and function in the thyroid gland and pancreas of mice were analysed.
The sub-cellular localization of a receptor like Taar1 determines its availability for putative ligands. Therefore, sub-cellular localization of Taar1 was analysed in thyroid tissue. Taar1 localization was confined to primary cilia. It is also noteworthy that sub-cellular localization of the Tg-processing protease, cathepsin L, was confined to primary cilia of thyrocytes. Thus, we were interested if an interaction of Taar1 with cathepsins takes place in the thyroid, and whether this affects the sub-cellular localization of Taar1. To this aim, sub-cellular localization of Taar1 was investigated in FRT cells treated with cathepsin inhibitors. Experiments revealed that Taar1 localization is altered upon inhibition of cathepsin activity.
Furthermore, we determined the phenotype of the thyroid gland of taar1-/- mice in comparison to WT controls. Serum thyroid hormone concentrations were slightly decreased upon Taar1 deficiency, which was correlated with decreased expression levels and proteolytic activities of cathepsins in the thyroid gland of taar1-/- mice vs WT controls. Thereby, taar1-/- mice were characterized as mildly hypothyroid, revealing the importance of Taar1 for regulation of thyroid gland functions.
Taar1 was shown previously to be also expressed in β cells of the pancreas. Thus, we aimed to investigate blood glucose concentrations upon Taar1 deficiency. However, the data showed that blood glucose levels were not affected by Taar1 deficiency. Collectively, this study revealed that Taar1 is important for various aspects of endocrine regulation in mice.
Supported by Deutsche Forschungsgemeinschaft in the framework of SPP 1629, BR1308/11-1 and 11-2
Development of a generalized process model for optimization of biotechnological processes
No full textComplex bioprocesses require effective process control strategies. Especially model based control strategies show a high potential for the optimization of bioprocesses. The mathematical models in these algorithms must fulfill two major requirements. First, the model must be able to describe the mechanistic relationship between actuating variables like feeding rates and the main state variables, like biomass and product concentrations. Second, the parameterized model must be able to predict the time course of the state variables of a cultivation process. Model development appears to be a time consuming and laborious task. In order to reduce the time necessary for model development, a new structured compartment model was developed, which is easy to adapt to different cultivation processes, as will be shown in this contribution.
The model developed is a new six-compartment model (6CM), which was used to describe the time course of batch and fed-batch cultivations of the bacteria Escherichia coli and Lactobacillus delbrueckii, the yeast Saccharomyces cerevisiae, the fungus Cyathus striatus and the cell line hybridoma.
The model is divided into the compartments primary biomass Xpri, product forming biomass Xp, structural biomass Xs, inactive primary biomass Xi, inactive structural biomass Xsi and dead biomass Xd. The primary biomass represents all reactions related to cell growth. This compartment is reproduced autocatalytically by carbon and nitrogen source uptake. The product producing biomass is responsible for secondary metabolite or induced protein (e.g. GFP) production. The formation of this compartment is catalyzed by the primary biomass. The growth of the compartment of structural biomass is also catalyzed by the primary biomass. Xs consists of membranes, cell walls and complex polysaccharides. It is formed by utilization of a carbon sources. Primary biomass and structural biomass may be inactivated to form inactive primary and structural biomass and may be reactivated to represent the maintenance metabolism. Under certain process conditions the inactive biomass compartments will be converted into dead biomass. Major properties of the model are the following:
1. The substrate concentration influences its uptake rate and therefore the rates of all further reactions.
2. Substrates are used for biomass and for product formation as well as for energy generation via fermentation or energy generation via cellular respiration.
3. Uptake rates, inactivation and death rates as well as yield coefficients are functions of the state variables and are modulated by double sigmoidal functions.
In order to adapt the model to a bioprocess a four-step modeling procedure was applied. First, a detailed process description was derived from experimental data and literature. Second, stoichiometric equations were formulated with respect to the metabolism under investigation. In the third step cause-effect relationship hypotheses, which were derived from process analysis, were used to identify the parameters of interest. Finally, these model parameters were estimated manually to adjust the model to the measured data.
The model is able to describe the time courses of important state variables, such as the concentrations of carbon and nitrogen, intermediate products, and products of primary and secondary metabolism as well as induced products. It is capable of describing O2 uptake und CO2 production via exhaust gas composition (aerobic processes) or gas flow rates (anaerobic processes). The model was adapted to each cultivation process only by choosing different values for the model parameters. This reduces the modelling effort for a new process significantly
Design and validation of a comprehensive model for risk-assessment in product development
No full textAbstract
Shorter development time of new products and a shift from complicatedness to com-plexity (see Glossary: complex vs. complicated) requires new thinking in product de-velopment.
To handle this trend there is a need for a better and more comprehensive knowledge of risks and challenges in product development. There are a number of risk evalua-tion methods available focused on different goals; e.g. project management, prod-ucts, organization.
The scope of this thesis is not to add another new method to the market but to create a practical approach handling risks and challenges in product development in a more comprehensive and integrated manner. All activities in this method, from the technology roadmap to the start of production, are based on common and approved methods; e.g. Failure Mode and Effect Analysis (FMEA), Value Analysis, Kano model.
The objectives are reducing lead-time in product development and to make schedul-ing more manageable. To reduce the lead-time a new and modified approach of the FMEA method is used. To increase the efficiency of the method, without any appre-ciable decrease of the effectiveness, risks, evaluated by severity and occurrence will omitted from further consideration below a certain threshold level.
A further increase of effectiveness and efficiency is expected from the strategy that the technique is based on common and approved methods, to initiate the method with a “flying start” and also to reduce the employee’s resistance against new meth-ods.
This thesis was carried out in cooperation with Dräger Safety, Lübeck, to set up a method for practical use which can be validated in practice.
This thesis is structured into 4 sections:
1. Basic principles of risk management
2. Description of structure and behavior of risk management handling in product development
3. Exemplary validation of the new risk evaluation in the context of the product development environment at Dräger Safety (in extract)
4. Discussion and outloo
Visualization and Heuristic Modeling for Planning of Minimally- and Non-Invasive Tissue Ablation
No full textThis PhD thesis describes methods for the support of the planning process for minimally- and non-invasive tissue ablation procedures. It focuses on the utilization of visualization and heuristic modeling to solve complex problems such as access path determination for needle-based tumor ablation therapies or sonication planning for high-intensity focused ultrasound.
For needle-based interventions, the manual selection of suited access paths is addressed, which typically requires the careful inspection of every candidate path as a whole to make sure that no structure at risk would be penetrated. Especially for angulated paths that are not included in one image slice, this results in repeated inspection of multiple slices. To improve this process, a visualization method for the highlighting of infeasible paths in common 2D viewers is proposed.
As an alternative to manual planning of percutaneous procedures, heuristic approaches to therapy plan optimization are investigated. An algorithm based on projection utilizing the GPU and image processing is proposed. Under consideration of multiple clinically relevant criteria, the method generates a list of optimal paths within a few seconds. A second, semi-automatic method is proposed, which represents a compromise between fully automatic and manual planning. It combines the fast projection based method with a numerical approach for Pareto-front approximation and allows for interactive exploration of the solution space.
To improve the manual planning of high-intensity focused ultrasound therapies, a real-time approximation of temperature and thermal dose is developed. It is based on numerical simulation for a range of exemplary configurations in a preprocessing step. During interactive planning, the temperature or thermal dose field for the situation at hand is interpolated and combined with an approximation of the heat sink effect resulting from vessels in proximity. The result is visualized in an interactive manner
Priorisierung präventiver Leistungen - Präferenzen aus Sicht der Bevölkerung zur horizontalen Priorisierung von Versorgungsbereichen sowie vertikalen Priorisierung präventiver Leistungen sowie den Priorisierungskriterien „Kosten“, „Nutzen“, „Zielgruppe“ und „Art der Prävention"
No full textThe study examined preferences amongst different health care areas and different preventative services. In addition, it investigated the importance of the prioritization criteria of costs, benefits, types of prevention and target groups for the benefit assessment of preventive services. Diverse demographic and health-related characteristics were analyzed as possible influencing factors on the attitudes of respondents.
An online survey was conducted with participants with a quota sampling based on sex, age and SES primarily through social networks in Germany. Rankings were used to examine the importance of 7 health care areas and 12 different preventive services. In the online survey, preference measurements were carried out using a Traditional-Conjoint-Analysis (TCA) and a Discrete-Choice-Experiment (DCE). The attributes costs, benefits, target groups and types of prevention were included, described by different factor levels. Participants were asked to give information about their sociodemographic and health-related characteristics. Data was analyzed using non and parametric-tests, OLS-regression and COX-regression.
244 people participated in the survey. In the opinion of respondents, the treatment of acute life-threatening injuries and diseases has the highest priority in the horizontal priority setting of health care areas, while secondary prevention follows in second, primary prevention in fourth and tertiary prevention was posteriorized. Medical-preventive services were clearly preferred to non-medical preventive services. The findings regarding the TCA show that benefits is the most important criterion for the respondents for the allocation of preventative services, followed by target groups, types of prevention and costs. In the DCE, priority was clearly given to the priority criteria benefits, followed by target groups, costs and types of prevention. There was hardly any association between the perceived preferences and the personal influencing factors
Overcoming Catastrophic Interference by Conceptors
No full textCatastrophic interference has been a major roadblock in the research of con- tinual learning. Here we propose a variant of the back-propagation algorithm, “conceptor-aided back-prop” (CAB), in which gradients are shielded by concep- tors against degradation of previously learned tasks. Conceptors have their origin in reservoir computing, where they have been previously shown to overcome catas- trophic forgetting. CAB extends these results to deep feedforward networks. On the disjoint MNIST task CAB outperforms two other methods for coping with catastrophic interference that have recently been proposed in the deep learning field
Automated Medical Image Processing Using Efficient Shape Descriptors: Principles and Applications
No full textThis thesis focuses on the development of fast and automated methods for detecting and segmenting anatomical structures in various modalities of medical imaging using efficient shape descriptors. The identification and delineation of these targeted objects are fundamental steps for further computer assisted applications, such as surgery planning, surgery interventional guidance, computer-aided detection and diagnosis or information fusion. First of all, a brief introduction of fundamental topics in medical image processing will be given, including segmentation, registration, detection and classification. Common techniques and methodologies used in these domains are generally reviewed. Then, a review of shape descriptors that are commonly used in medical image processing is conducted. The review will categorize these techniques with respect to different dimensions, such as complexity, efficiency, degree of user interactions and sensitivity to parameters, etc. Afterwards, to demonstrate how these shape descriptors are applied in each individual clinical task, several segmentation, registration, classification and detection tasks, where a variety of shape descriptors serve as the mainstay, will be described in detail. Specifically, the tasks consist of the segmentation of femur heads in fluoroscopic images using a Gabor-based Hough shape descriptor, the segmentation and registration of breasts in magnetic resonance images, the detection of nipples in 3D breast ultrasound images and the segmentation of liver vessels in multi-phase computer tomography images using a variety of Hessian-based shape descriptors. Meanwhile, a computer-assisted diagnostic tool dedicated to breast lesion classification is proposed, on the basis of a series of sphere packing shape descriptors. For each task, clinical background will be first explained, and the state-of-the-art techniques that attempted to resolve the problem will be reviewed
Linking environmental change, preferences and migration in coastal areas of Ghana and Indonesia
No full textCoastal regions are associated with large and growing concentrations of human populations, driven especially by economic growth and in-migration. This rapid coastal development contributes significantly to coastal degradation and, at the same time, leads to an increased exposure of a high number of people to increasing threats of coastal hazards, which holds especially true for the developing country context (Nicholls et al. 2007; Neumann et al. 2015). Therefore, this dissertation tries to shed some light on linkages between migration and coastal environments in two coastal regions in Ghana and Indonesia.
More precisely, research 1 addresses the question whether coastal environmental threats are drivers of out-migrations from the two study regions, which experience serious coastal changes like shoreline erosion, land subsidence and recurring floods. A household survey with migrants and non-migrants has been conducted and measures of these rather gradual coastal changes—ranging from individual perceptions to expert categorizations—have been collected.
Research 2 tries to explore which other factors are linked to migration decisions to understand how to anticipate migration flows in those regions. It especially focuses on individual time and risk preferences as they are theoretically very likely to influence individual migrations (and also the link between the environment and migration) but have not been much tested empirically.
Research 3 addresses the question whether migrants differ from non-migrants in their behavior toward the coastal environment using two promising proxies for actual behavior. The first addresses underlying attitudes toward coastal environments, while the other one focuses on cooperative behavior in a common-pool resource experiment.
All three researches combined do not only advance methodological aspects within the migration literature but also help to shed some light on migration issues in coastal regions