In this work I present conclusions of clinical-laboratory research focused on the patients with diabetic macular edema (DME). We performed biochemical and immunochemical analyses of vitreous samples that were collected during the pars plana vitrectomy. Moreover, at patients with non-proliferative diabetic retinopathy (NPDR) we assessed morphological characteristics of DME using optical coherence tomography (OCT). According to our findings, the vitreous and serum concentrations of uric acid and glucose were significantly higher in patients with diabetic retinopathy and DME compared to controls. Also total ratio (serum/ vitreous concentration) of uric acid and glucose was in diabetics significantly higher than in controls. The most important determinant of increasing concentration of both uric acid and glucose in the vitreous was the grade of diabetic retinopathy. Moreover, we demonstrated significant correlation between vitreous concentration of uric acid and concentration of the vascular endothelial growth factor (VEGF) in patients with DME and NPDR. We found further, that the volume of the macula (cube volume - CV) computed with the software of Cirrus HD-OCT correlates in diabetics significantly with the vitreous VEGF concentration, but not with uric acid. This OCT parameter could be used to...

This diploma thesis includes an experimental and a bioinformatic part. The two parts are linked together through the subject of transcription factors of 'forkhead box O' (FOXO) family. FOXO transcription factors have a key role in many cellular processes including cell cycle regulation, apoptosis and metabolism. For a long time, they have been considered strictly as the tumor-suppressors yet a growing number of evidence is pointing out to their pro-tumorigenic role. In consequence FOXO transcription factors are studied intensively as potential therapeutic targets in cancer. In the past decade, in silico prediction of protein-protein interactions has become popular in basic research as well as in drug development. Nonetheless, the predicted structures are still far from fitting to the expected behavior of the respective biomolecules. In the experimental part of this thesis, I verified the interaction of four in silico predicted protein binders based on naturally occurring PDZ domain with FOXO4 using microscale thermophoresis. Non-invasive bladder tumors represent a heterogeneous disease where reliable prediction of tumor aggressiveness is still lacking despite an intensive research. In the bioinformatic part of this thesis, I described the cellular composition of the tumor microenvironment and demonstrated...

6 Abstract Transcription factors play a key role in the management of cell growth and differ- entiation and their deregulation is associated with many cancers. TEAD proteins utilise highly conserved DNA binding domain to recognise specific DNA sequences. This domain could facilitate new drug design and development. The goal of this master thesis includes recombinant preparation of DNA binding domain of transcriptional factor TEAD4 extended by a part of an unstruc- tured variable sequence, which connects this domain with transactivation domain. Purification steps include affinity chromatography followed by size exclusion chro- matography. The characterization of produced protein was performed by mass spectrometry and finally, native gel electrophoresis was used to prove the ability of the produced protein to bind DNA. During purification steps, a fragmentation from C-terminus was observed. Based on analysis of the mass spectra, three most represented forms of produced protein were described all of which were fragmented. The most abundant form (55%) consisted of amino acids 30-131 from TEAD4 protein. Second most abun- dant form (18%) consisted of amino acids 30-144 and the third form consisted of amino acids 30-81. Native gel electrophoresis verified the ability to bind DNA, the efficiency was however lower...

Polyketides are natural products with complex chemical structures and immense pharmaceutical potential that are synthesized via secondary metabolic pathways. The in-vitro synthesis of these molecules requires high supply of building blocks such as acetyl Co-enzyme A, and cofactors (adenosine triphosphate (ATP). These precursor and cofactor are synthesized from respective soluble enzymes. Owing to the expensive nature of the enzymes, it is important to immobilize enzymes to improve the process economics by enabling multiple uses of catalyst and improving overall productivity and robustness. The polymer-based particles of nano and submicron size have become attractive material for their role in the life sciences. With the advances in synthetic protocols of the microgels and commercial availability of many of the monomers, it is feasible to tune the properties of the particles as per the process requirement. The core shell microgel with functional shell allows high loading of ligands onto the microgel particles due to increased availability of functional group on the outer surface. The aim of the thesis thus was to study biochemical reactions on the smart microgels support using single (acetyl CoA synthetase (Acs)) and dual (pyruvate kinase (Pk) and L-lactic dehydrogenase (Ldh)) enzyme/s systems. The study indicated that the enzyme immobilization significantly depends on the enzyme, conjugation strategy and the support. The covalent immobilization provides rigidity to the enzyme structure as in case of Acs immobilized on PNIPAm-AEMA microgels but at the same time leads to loss in enzyme activity. Whereas, in the case of covalent immobilization of Ldh on microgel showed improved in enzyme activity. On the other hand adsorption of the enzyme via ionic interaction provide better kinetic profile of enzymes hence the membrane reactor was prepared using PNIPAm-PEI conjugates for acetyl CoA synthesis. The better outcome of work with PNIPAm-PEI resulted in its further evaluation for dual enzyme system. This work is unique in the view that the immobilization strategies were well adapted to immobilize single and dual enzymes to achieve stable bioconjugates for their respective applications in precursor biosynthesis (Acetyl Co enzyme A) and co-factor dependent processes (ACoA and ATP). The positive end results of microgels as the support (particles in solution and as the thin film (membrane)) opens further prospective to explore these systems for other precursor biomolecule production.

Die geochemischen Verhältnisse der Böden des Erzgebirge und Vogtlandes werden auf der Grundlage der komplexen geochemischen Analyse von ausgewählten, flächenrepräsentativen Bodenprofilen auf Hauptgesteinen des Untersuchungegebietes charakterisiert. Die Wirkungsweisen vorhandener Einflussfaktoren (Gesteinschemismus, Bodengenese, Bodennutzung) werden untersucht und bewertet. Die Untersuchungen stellen eine Grundlage für die Ableitung von geologisch bedingten regionalen Hintergrundbelastungen dar. / A characterization of the soil geochemistry of the Erzgebirge and Vogtland Regions (Saxony) is provided on the basis of the investigation of characteristic soil profiles on major bedrock types of the area. Different factors of influence (rock geochemistry, soil formation, land utilization) are investigated and evaluated. The investigation provides essential information for the development of regional background concentrations.

Major accidents in the process industries occurred mostly as an outcome of multiple failures in different safety barriers and their interrelation with unsafe acts by frontline operators. This has become the reason why safety analyses in terms of plant technical aspects cannot be performed independently from analysing human response to the changing technology. Unsafe acts and errors by operators must be seen as a symptom of system insufficiencies and underlying problems, rather than as the cause of an accident. With this paradigm, the need to optimally configure the system and the whole working condition to understand human’s limitation and requirements becomes very evident. It is too naive to desire that human operators make zero error by asking them to change their behaviour and to perfectly adapt to the system. Human Factors (HF) attempts to cope with the need to understand the interrelation between human operators, the technology they are working with and the management system, with the aim to increase safety and efficiency. In achieving this goal, HF must be incorporated into the whole plant lifecycle, from the earliest design stage to plant operation and modifications. Moreover, HF analysis must comprise all kinds of operators’ activities and responsibilities in operating process plants, which can include manual works in field and supervisory control conducted remotely from a control centre/room. This work has developed techniques that provide systematic way to incorporate HF into process plant lifecycle. The new HF analysis technique, PITOPA-Design, in a combination with the classic PITOPA, is applicable for an implementation during design and operation of a plant. With the awareness that safety analysis and HF cannot be performed separately, an interconnection with HAZOPs is made possible by means of this new technique. Moreover, to provide a systematic analysis of operators’ work in control room, an additional technique, the PITOPA-CR was also developed. This HF technique can as well be integrated into a general HF analysis both during design phase and plant operation. In addition to it, results coming from PITOPA-CR will provide information required to optimally configure control and alarm system, as well as the whole alarm management system to better understand the limitation and requirements of control room operators. The structure of the development can be described as follows: i) Development of HAZOPA (the Hazards and Operator Actions Analysis), which provides the interconnection between HF analysis and HAZOPs, ii) Development of PITOPA-Design, a technique to incorporate HF consideration into design phase, which is differentiated into 3 stages to comprise the conceptual design, the basic engineering and the detail engineering phase, iii) Development of PITOPA-CR, a technique for HF analysis in control room, iv) Integration of PITOPA-CR into alarm management system, development of a technique for alarm prioritization.:ACKNOWLEDGEMENT i ABSTRACT iii ZUSAMMENFASSUNG iv CONTENTS v TABLE OF FIGURES viii LIST OF TABLES x NOMENCLATURE xi ACRONYMS AND ABBREVIATIONS xii CHAPTER 1 1 INTRODUCTION 1 1.1 Background 1 1.2 Objectives 2 1.3 Scope of Work 3 CHAPTER 2 5 THEORETICAL BACKGROUND 5 2.1 Fundamentals of Human Error 5 2.2 Human Factors (HF) 8 2.3 Motivations to Consider HF in Process Safety 9 2. 3. 1 Accidents that Address HF in Process Safety 11 2. 3. 2 Regulation and Legal Requirements 16 2. 3. 3 Business Value 19 2.4 Work of Operators in Complex Systems 19 2. 4. 1 Role of Operators in Complex Systems 20 2. 4. 2 Problems with Computerisation and Automation 24 2. 4. 3 Allocation of Functions and Levels of Automation 25 2.5 Performance Influencing Factors (PIFs) 27 2.6 Distributed Control System (DCS) and Alarm Systems 29 2. 6. 1 Alarm, Alarm System and Alarm Management 30 2. 6. 2 Most Common Alarm Problems 33 2. 6. 3 Improving Alarm Performance through Prioritization 34 2.7 Safety Analysis Methods 38 2.7.1 Qualitative Safety Analysis 39 2.7.2 Quantitative Safety Analysis 43 2.8 Mathematical Algorithms 44 2.8.1 Techniques for Multi-Criteria Decision Making (MCDM) 44 2.8.2 Classification Methods 47 CHAPTER 3 50 RECENT DEVELOPMENTS IN HF STUDIES 50 3. 1 Methods for HF analysis 50 A. Task Analysis 50 B. Techniques for Operators Actions Analysis 51 3. 2 Human Reliability Analyses (HRA) 52 3. 3 Consideration of Human Error in HAZOP 53 3. 4 HF in Process Plant Design 54 3. 5 HF in Alarm Management and DCS-Design 55 3. 6 The Need for Further Development of HF Methods 57 CHAPTER 4 58 MOTIVATION OF THE WORK 58 CHAPTER 5 61 PROCESS INDUSTRY TOOL FOR OPERATOR ACTIONS ANALYSIS (PITOPA) 61 5.1 The New Technique for Operator Actions Analysis (OAA) 64 5.2 Technique for Performance Influencing Factors (PIFs) Evaluation 65 5.3 Validation of PITOPA in the Process Industry 67 CHAPTER 6 71 EXTENDING HAZOP TO INTEGRATE HF INTO 71 GENERAL SAFETY ANALYSIS 71 6.1 Development of HAZOPA (The Hazard, Operability and Operator Actions Analysis) 72 6.2 Case Study 75 CHAPTER 7 85 APPROACH TO INCORPORATING HF CONSIDERATION 85 INTO PLANT DESIGN 85 7.1 Development of an Approach for HF Analysis in Design – The PITOPA-Design 85 7.1.1 HF Analysis in Conceptual Design Phase (HFAD–Conceptual) 88 7.1.2 HF Analysis in Basic Engineering (HFAD – Basic) 93 7.1.3 HF Analysis in Detail Engineering (HFAD-Detail) 107 7.2 Technique for HF-Design Parameters Evaluation 109 7.3 Intermediate Summary 114 CHAPTER 8 115 IMPLEMENTATION OF THE NEW PITOPA-DESIGN: 115 A CASE-STUDY 115 8.1 Conceptual Design 115 8.2 Basic Engineering 123 8.3 Detail Engineering 127 CHAPTER 9 132 APPROACH FOR IMPROVING OPERATOR PERFORMANCE 132 IN CONTROL ROOM 132 9.1 Performance Influencing Factors (PIFs) for Supervisory & Monitoring Tasks 134 9.2 Development of PITOPA-Control Room (PITOPA-CR) 140 9.2.1 Analysis of Normal Operation 142 9.2.2 Analysis of Abnormal Operation 150 9.3 Alarm Prioritization 156 9.3.1 A survey on Alarm Prioritization 156 9.3.2 Incorporation of CROAA into Alarm Prioritization 157 9.4 Intermediate Summary 165 CHAPTER 10 167 INCORPORATION OF OPERATOR ACTIONS ANALYSIS INTO ALARM MANAGEMENT 167 CHAPTER 11 171 RESULTS AND FUTURE WORKS 171 11. 1 Results 171 11. 2 Future Works 172 BIBLIOGRAPHY 174 APPENDIX A A-1 APPENDIX B B-1 / Schwere Unfälle in der Prozessindustrie erfolgen meist aus einem Zusammenspiel mehrerer verschiedener Fehler und der gleichzeitigen Wechselwirkung mit falschem menschlichem Handeln. Dabei sind diese Fehlhandlungen nicht als Unfallursache anzusehen, sondern sie resultieren aus Fehlern, die in dem System selbst zu finden sind. Aus diesem Grund kann bei der Sicherheitsanalyse die technische Analyse nicht unabhängig von der Betrachtung des Human Factors (HF) durchgeführt werden. Um eine Reduzierung der Fehlhandlungen zu erreichen, müssen das Anlagendesign, die Bedienbarkeit und die Arbeitsumgebung an die menschlichen Fähigkeiten angepasst werden. Human Factors (HF) betrachtet die Interaktion zwischen menschlichen, technischen und organisatorischen Aspekten einer Anlage, mit dem Ziel die Sicherheit und Effektivität der Anlage zu optimieren. Dafür ist eine Einbindung von HF in den gesamten Lebenszyklus einer Anlage notwendig. So müssen HF- Analysen nicht nur während des Betriebs einer Anlage und bei Prozessmodifikationen durchgeführt werden, sondern auch während des gesamten Design- Prozesses, da gerade in den frühen Design-Phasen das Optimierungspotential besonders hoch ist. Eine solche Analysemethode muss alle Aufgaben eines Operators erfassen, so dass zwischen manueller Arbeit und der Arbeit in der Leitwarte unterschieden werden muss. In dieser Arbeit wurden Analysentechniken entwickelt, die einen systematischen Ansatz zur Berücksichtigung des HF über den gesamten Lebenszyklus einer verfahrenstechnischen Anlage darstellen. Mit Hilfe der neuen Analysemethode, PITOPA-Design, können Untersuchungen sowohl während der Designphase als auch während des Betriebs einer Anlage durchgeführt werden. Da solche HF-Analyse immer in Verbindung mit einer klassischen Sicherheitsanalyse erfolgen muss, bindet die neue Methode die HAZOP-Analyse direkt ein. Darüber hinaus wurde ein weiterer Ansatz für die Analyse von Operatorhandlungen in einer Messwartenarbeit entwickelt. Diese neue Analysentechnik, PITOPA-CR, bildet die Grundlage für Verbesserungen im Alarmsystem und wird in das Alarmmanagementsystem eingebunden. Die Arbeit ist wie folgt strukturiert: i) Entwicklung von HAZOPA (the Hazards and Operator Actions Analysis). Diese Methode stellt die Einbindung der HF-Analyse in HAZOP dar. ii) Entwicklung von PITOPA-Design, zur HF-Analyse während des gesamten Designprozesses einer verfahrenstechnischen Anlage. Die Methode wurde in 3 Teile eingeteilt, um die drei Designsphasen Conceptual-, Basic-, und Detail-Design zu erfassen. iii) Entwicklung von PITOPA-CR, zur HF-Analyse in der Messwarte. iv) Einbindung von PITOPA-CR in das Alarmmanagementsystem und Entwicklung einer Technik zur Alarmpriorisierung.:ACKNOWLEDGEMENT i ABSTRACT iii ZUSAMMENFASSUNG iv CONTENTS v TABLE OF FIGURES viii LIST OF TABLES x NOMENCLATURE xi ACRONYMS AND ABBREVIATIONS xii CHAPTER 1 1 INTRODUCTION 1 1.1 Background 1 1.2 Objectives 2 1.3 Scope of Work 3 CHAPTER 2 5 THEORETICAL BACKGROUND 5 2.1 Fundamentals of Human Error 5 2.2 Human Factors (HF) 8 2.3 Motivations to Consider HF in Process Safety 9 2. 3. 1 Accidents that Address HF in Process Safety 11 2. 3. 2 Regulation and Legal Requirements 16 2. 3. 3 Business Value 19 2.4 Work of Operators in Complex Systems 19 2. 4. 1 Role of Operators in Complex Systems 20 2. 4. 2 Problems with Computerisation and Automation 24 2. 4. 3 Allocation of Functions and Levels of Automation 25 2.5 Performance Influencing Factors (PIFs) 27 2.6 Distributed Control System (DCS) and Alarm Systems 29 2. 6. 1 Alarm, Alarm System and Alarm Management 30 2. 6. 2 Most Common Alarm Problems 33 2. 6. 3 Improving Alarm Performance through Prioritization 34 2.7 Safety Analysis Methods 38 2.7.1 Qualitative Safety Analysis 39 2.7.2 Quantitative Safety Analysis 43 2.8 Mathematical Algorithms 44 2.8.1 Techniques for Multi-Criteria Decision Making (MCDM) 44 2.8.2 Classification Methods 47 CHAPTER 3 50 RECENT DEVELOPMENTS IN HF STUDIES 50 3. 1 Methods for HF analysis 50 A. Task Analysis 50 B. Techniques for Operators Actions Analysis 51 3. 2 Human Reliability Analyses (HRA) 52 3. 3 Consideration of Human Error in HAZOP 53 3. 4 HF in Process Plant Design 54 3. 5 HF in Alarm Management and DCS-Design 55 3. 6 The Need for Further Development of HF Methods 57 CHAPTER 4 58 MOTIVATION OF THE WORK 58 CHAPTER 5 61 PROCESS INDUSTRY TOOL FOR OPERATOR ACTIONS ANALYSIS (PITOPA) 61 5.1 The New Technique for Operator Actions Analysis (OAA) 64 5.2 Technique for Performance Influencing Factors (PIFs) Evaluation 65 5.3 Validation of PITOPA in the Process Industry 67 CHAPTER 6 71 EXTENDING HAZOP TO INTEGRATE HF INTO 71 GENERAL SAFETY ANALYSIS 71 6.1 Development of HAZOPA (The Hazard, Operability and Operator Actions Analysis) 72 6.2 Case Study 75 CHAPTER 7 85 APPROACH TO INCORPORATING HF CONSIDERATION 85 INTO PLANT DESIGN 85 7.1 Development of an Approach for HF Analysis in Design – The PITOPA-Design 85 7.1.1 HF Analysis in Conceptual Design Phase (HFAD–Conceptual) 88 7.1.2 HF Analysis in Basic Engineering (HFAD – Basic) 93 7.1.3 HF Analysis in Detail Engineering (HFAD-Detail) 107 7.2 Technique for HF-Design Parameters Evaluation 109 7.3 Intermediate Summary 114 CHAPTER 8 115 IMPLEMENTATION OF THE NEW PITOPA-DESIGN: 115 A CASE-STUDY 115 8.1 Conceptual Design 115 8.2 Basic Engineering 123 8.3 Detail Engineering 127 CHAPTER 9 132 APPROACH FOR IMPROVING OPERATOR PERFORMANCE 132 IN CONTROL ROOM 132 9.1 Performance Influencing Factors (PIFs) for Supervisory & Monitoring Tasks 134 9.2 Development of PITOPA-Control Room (PITOPA-CR) 140 9.2.1 Analysis of Normal Operation 142 9.2.2 Analysis of Abnormal Operation 150 9.3 Alarm Prioritization 156 9.3.1 A survey on Alarm Prioritization 156 9.3.2 Incorporation of CROAA into Alarm Prioritization 157 9.4 Intermediate Summary 165 CHAPTER 10 167 INCORPORATION OF OPERATOR ACTIONS ANALYSIS INTO ALARM MANAGEMENT 167 CHAPTER 11 171 RESULTS AND FUTURE WORKS 171 11. 1 Results 171 11. 2 Future Works 172 BIBLIOGRAPHY 174 APPENDIX A A-1 APPENDIX B B-1

The work of an engineer is closely intertwined with safety. An engineer’s perception of the “safety” task is traditionally inherent in his or her design. However, in the technical world most machines and systems designed by engineers contain a human element, which engineers have to consider in their work. In the oil and gas upstream industry – especially drilling, production and workover operations – petroleum engineers (including drilling and production engineers) are responsible not only for design but also for operational and organizational aspects. The human factor becomes more important in complex offshore operations. Incorporating safety into a sys-tem design requires identifying, analyzing and evaluating risks and ensuring that any not accounted for are taken into consideration. This process requires communication among everyone involved in the process. Analysis of accidents in the oil and gas industry shows that often a lack of that communication led to incident triggering events. In this thesis, the author proposes a novel communication model that improves this exchange of information and supposedly makes the process of risk assessment more effective. In addition, the new model also incorporates factors such as emotions, feelings, needs and imagination into the risk assessment process. This broadens the information base for the risk identification and analysis and creates an atmosphere of psychological ownership for the stakeholders in the process, which leads to a perceived safety climate in the organization where the new model is applied. The innovative communication or consultation model, as it is also referred to in risk assessments, is based on a structured process used in conflict resolution called mediation. Mediation is an alternative conflict resolution process that is centered on mutual under-standing and listening to each other’s needs. The process is composed of elements that characterize it. These elements of mediation are used to assess other communication processes and to develop new communication models. The application of the elements of mediation and the safety-mediation consultation into the risk assessment process enables this process to be enhanced with human factors such as emotions, feelings, intuition and imagination. The inclusion of all stakeholders creates psychological ownership, improves communication, enables organizational learning and expands the knowledge base for risk analysis. The applicability of the safety-mediation consultation process for a human factor-based risk assessment is presented and tested using illustrative examples and field cases from the international oil and gas industry. Possible concerns and limitations are also discussed. This thesis shows that mediation and elements of the mediation process can be applied to improve communication in the international oil and gas industry. This is facilitated by educated safety mediators, who help the petroleum engineer and operational crew on a drilling rig to achieve a better understanding by ensuring that they hear and fully register each other’s needs.

This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance. Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology. The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells. Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system. Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.

SMOFLipid® is a commonly used fat emulsion for parenteral nutrition. We investigated how enrichment of SMOFLipid® with n-3 polyunsaturated fatty acids (PUFA) in a form of second fat emulsion, Omegaven® , changes fatty acid composition of total plasma phospholipids and erythrocyte phospholipids, cytokine concentrations in serum and in supernatant from in vitro whole blood culture stimulated with lipopolasaccharide (LPS) and we evaluated also changes in oxido- reductive balance. Eight patients on long-term home parenteral nutrition recieved both emulsions, SMOFLipid® (6 weeks) and SMOFLipid® +Omegaven® (4 weeks), one by one. We observed no significant differences in common laboratory and clinical parameters between these two types of diet. Enrichment of SMOFLipid® with Omegaven® led to an increase in eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) in total plasma phospholipids and there was also an increse in proportion of EPA in erythrocyte phospholipids, while proportion of DHA remained unchanged. These changes were in both phospholipids of plasma and erythrocyte compensated for a decrease in proportion of linoleic and arachidonic acid (n-6 PUFA). There were elevated IL-6 and TNF-α serum concentrations in patients after both diets. There was a decrease in IL-6 production by 36% with SMOFLipid®...

The pathogenesis of insulin resistance is a complex and still intensively studied issue. Endocrine and paracrine activity of the adipose tissue together with mi- tochondrial dysfunction are the most discussed potential factors included in the development of insulin resistance. In the first part of our study we examined the involvement of the adipose tissue and its secretory products in the etiopathogenesis of insulin resistance in patients with Cushing's syndrome, acromegaly and simple obesity. We focused on three important regulators of metabolic homeostasis - fibroblast growth factors 21 and 19 (FGF-21 and FGF-19) and adipocyte fatty acid binding protein (FABP-4). We found significantly elevated circulating levels of FGF-21 and FABP-4 ac- companying insulin resistance in both patients with simple obesity and patients with obesity connected to Cushing's syndrome, as compared to healthy controls. The concentrations of both substances were comparable between hypercortisolic and obese patients. This finding together with the absence of correlation be- tween the levels of FGF-21 resp. FABP-4 and cortisol suggest that the reason for elevation of their concentrations is obesity and its metabolic consequences themselves rather then the effect of hypercortisolism on FGF-21 and FABP-4 production. We found no...