Mesomorphism, molecular structure and dynamics of polydiethylsiloxane

The correlation between the phase behaviour of polydiethylsiloxane (PDES) and conformational and motional changes at the various disordering transitions has been investigated by nuclear magnetic resonance (n.m.r.), dielectric relaxation and shear experiments. Diffusive rotation of the chain segments around the long axis of the molecules is indicated by 29Si n.m.r. below the isotropization transition. The remarkably fluid character is explained in part by the coexistence of anisotropic and isotropic motional states of the -OSiEt2- segments, which indicate microscopic domain formation. The molecular motion in the α2- and β2-phases is restricted but still fast with respect to the 29Si n.m.r. timescale. Transmission electron micrographs show, besides chain-folded lamellae, also extended-chain lamellae. These differences in the morphologies can explain why the interconversion of α2-PDES into the thermodynamically more stable β2-polymorph is slow, in spite of the pronounced mobility of the polymer segments. Long-range reorganization processes have to be considered to allow the morphological changes observed by electron microscopy

Advanced Raman Spectroscopy for Bioprocess Monitoring:Dissertation

The Raman -effect was discovered almost 90 years ago. It took a long time until the importance of Raman spectroscopy was fully understood and accepted for process industrial applications. Still today the usage is limited to a small application area. During the last decades advances in the production of spectroscopic components have reduced the complexity of the instrumentation and further mediated a persistent decline in costs, thus making this technology available for a broader audience. The basic components of a Raman spectrometer are a monochromatic light source, typically laser, a Raman probe with optical fibres, a spectrograph and a detector which is connected to a measurement- and control PC. Raman spectroscopy is increasingly becoming a choice as analytical tool in bioanalytics. Most of biological samples are handled in aqueous form which challenges many other analytical techniques (e.g., infrared spectroscopy). Raman and its enhancement techniques are able to measure quality and quantity of compounds in liquid phase with no or very little interference of water. The quantity of a compound can be determined by the peak-intensity and the quality by the position in the measured spectrum. Another advantage is that Raman spectroscopy does not rely on extensive sample preparation and measurements can be carried out non-invasively by placing an immersion probe with fibre optics directly in the liquid media. A Raman measurement is conducted fast, within milliseconds, and multiple relevant process parameters from the same sample can be measured at the same time. The measurements can be performed in continuous mode, i.e. one after the other or with a delay in-between. The operator can determine the measurement interval and in this way the development of a process can be observed online and in real-time. Further important advantages of spectroscopic methods over many other biochemical and physical measurement tools are the robustness as they do not require assays and they are rather unsusceptible against variations of pH, temperature changes, vibrations and other process parameters. If there is no coating, colour or special treatment of the glass, some Raman set-ups allow measurements directly through the glass into the liquid phase. This option enables real process measurements without the need to disturb or contaminate the analytes. These experimental set-ups are in the focus of this thesis. Despite great advantages of spectroscopic methods, the utilization is often complicated since the threshold values are often above what is required for screening. Besides the lack of sensitivity of conventional Raman in bioprocess applications, the major drawback of this technique so far has been the disturbance of the broad fluorescence background especially in biological samples. The main objective of this thesis was to find solutions for increasing the limit of detection (LOD) for biomolecules, being capable to detect them during the course of the process reliably and being able to diminishing background signals induced by sample- and matrix-related auto-fluorescence. The proposed solutions are mainly surface enhanced Raman spectroscopy (SERS), time-gated (TG) Raman spectroscopy and the combination of both. This thesis had a rather broad scope ranging from biofilm detection on water membrane filtration processes, over low-concentration bacteria detection with various enhancement options to finally follow the course of cell culture media development during cultivations. The aim of the thesis is to show that in challenging bioprocess-environments, Raman spectroscopy can detect weak signals over the background-noise from fluorescence in combination with SERS-enhancement-sensor techniques and with the time-gated Raman technology in particular.Der Raman-Effekt wurde schon vor fast 90 Jahren entdeckt. Anerkennung und Akzeptanz dieser Technologie für den Einsatz in der Verfahrenstechnik brauchten eine lange Zeit, wobei derzeit die Anwendungsbereiche noch sehr beschränkt sind. In den letzten Jahrzehnten haben technologische Fortschritte in der Produktion von optischen Komponenten, insbesondere von Lasern und Detektoren, die Komplexität von Raman-Spektrometern erheblich reduziert. Kostenreduzierung von Komponenten und Vereinfachung der Instrumente ermöglicht jedoch ein breiteres Anwenderfeld. Die Hauptkomponenten eines Raman-Spektrometers sind eine monochromatische Lichtquelle, typischerweise Laser, eine Raman-Sonde mit Lichtleitern, ein Spektrograph und ein Detektor, der mit einem Mess- und Steuer-PC verbunden ist. Die Raman-Spektroskopie wird zunehmend als analytisches Instrument in der Bioanalytik eingesetzt. Die meisten biologischen Proben werden in wässriger Form behandelt, was viele andere analytische Techniken (z.B. Nahinfrarotspektroskopie) herausfordert. Raman und insbesondere die oberflächen-verstärkte Raman-Spektroskopie (SERS) sind in der Lage, organische und anorganische Verbindungen in flüssigen Proben ohne großen Einfluss der Wasserbanden zu charakterisieren. Unter konstanten Messbedingungen ermöglichen Raman und SERS auch die Quantifizierung von Komponenten in der Probe. Hauptsächlich ermöglicht SERS aber die Bestimmung von Komponenten in sehr geringen Stoffkonzentrationen. Die Quantität eines Stoffes kann durch die Peak-Intensität und die Qualität durch die Position der Peaks im gemessenen Spektrum ermittelt werden. Ein weiterer Vorteil ist, dass die Raman-Spektroskopie nicht eine umfangreiche Probenvorbereitung benötigt und Messungen nicht-invasiv durchgeführt werden können. Eine faseroptische Raman-Sonde (spezielle Tauchsonde) kann auch für den Einsatz direkt in flüssigen Medien benutzt werden. Eine Raman-Messung kann sehr schnell (innerhalb von wenigen Millisekunden) durchgeführt und es können mehrere relevante Prozessparameter gleichzeitig bestimmt werden. Die Messungen können kontinuierlich, nacheinander oder in bestimmten Zeitintervallen durchgeführt werden. Auf diese Weise kann die Entwicklung eines Prozesses online und in Echtzeit beobachtet werden. Weitere wichtige Vorteile spektroskopischer Methoden gegenüber vielen anderen biochemischen und physikalischen Messwerkzeugen sind die Robustheit, da sie keine speziellen Assays erfordern, und sie sind eher unempfindlich gegenüber Variationen von Prozessparametern wie z.B. pH, Temperaturänderungen, Druck und Vibrationen. Falls es keine Beschichtungen, Einfärbungen oder spezielle Behandlungen des Glases gibt, erlauben einige Raman-Aufbauten Messungen direkt durch Glas in das flüssige Medium, z.B. durch Glasbehälter oder Küvetten. Diese Optionen ermöglichen reale Prozessmessungen von Proben ohne die Notwendigkeit die Analyten zu stören oder zu kontaminieren. Neben Durchflusszellen stehen diese experimentellen Aufbauten im Fokus dieser Arbeit. Trotz großer Vorteile der Raman-Spektroskopie, ist die Realisierung robuster Messergebnisse oftmals kompliziert, da die Schwellenwerte der Stoffkonzentrationen von den meisten Analyten in Bioprozessen oft unterhalb der Nachweisgrenze liegen. Neben dem Mangel an hoher Empfindlichkeit von konventionellem Raman bei Bioprozessanwendungen war der größte Nachteil dieser Technik bisher die Störung des breiten Fluoreszenzhintergrundsignals, welches das eigentliche Raman-Signal zum Teil stark überlagert. Das Hauptziel dieser Arbeit war es, Lösungen für die Verbesserung der Nachweisgrenze (Limit of Detection, LOD) für Biomoleküle zu finden, um in der Lage zu sein, den Prozessverlauf und einzelne Organismen zuverlässig charakterisieren zu können. Die vorgeschlagenen Lösungen sind vor allem die oberflächenverstärkte Raman-Spektroskopie (SERS), die zeitaufgelöste (time-gate, TG) Raman-Spektroskopie und die Kombination beider. Diese Doktorarbeit bearbeitet ein recht breites Feld von Anwendungen, angefangen von Biofilm-Erkennung auf Filtermembranen bei der Trinkwasseraufbereitung, über die Erkennung von Bakterien in sehr geringen Konzentrationen mit verschiedenen SERS Nanopartikeln und Oberflächensubstraten bis zur Messungen der Zusammensetzung von Zellüberstandproben während einer kompletten Kultivierung. Das Ziel dieser Arbeit ist es, in unterschiedlichen Einsatzfeldern von Bioprozessen zu zeigen, wie weiterentwickelte Raman-Spektroskopische Methoden eingesetzt werden können, um die sehr geringen biochemischen Signale vom störenden Einfluss der Hintergrundsignale, insbesondere der Fluoreszenz, unterscheiden und charakterisieren zu können

Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS)

Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.Peer reviewe

Mindfulness in informal caregivers of palliative patients

AbstractObjectives:Mindfulness is a concept of growing impact on psychotherapy and has been shown to be effective for stress reduction and to improve psychological well-being. Existential Behavioural Therapy (EBT) was developed to support relatives of palliative care (PC) patients to cope with their situation during caregiving and bereavement. Mindfulness training was a core element of the intervention.We investigated the relationship between mindfulness, mental distress, and psychological well-being in informal caregivers, and evaluated if the effects of the intervention were mediated by mindfulness.Methods:Relatives of PC inpatients took part in a randomized-controlled EBT trial and completed the Cognitive and Affective Mindfulness Scale-Revised, items from the Five Facets of Mindfulness as well as the Brief Symptom Inventory, the Satisfaction with Life Scale, the WHOQOL-BREF, a numerical rating scale on quality of life (range 0–10), and the Schedule for Meaning in Life Evaluation at pre- and post-intervention, and a 3- and 12-months follow-up.Results:One-hundred-and-thirty carers were included, most of them (71.6%) recently being bereaved at the beginning of the intervention. High correlations between mindfulness and mental distress (r = −0.51, p &lt; 0.001) as well as life satisfaction (r = 0.52, p &lt; 0.001) were found. Mindfulness was a significant predictor of improvement in psychological distress, meaning in life and quality of life three months after the intervention. The EBT effects were partly mediated by mindfulness.Significance of results:Mindfulness seems to be a promising concept in supporting informal caregivers of PC patients. Further research is needed to identify the required format and intensity of mindfulness practice necessary for improvement.</jats:sec

Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system

<p>Abstract</p> <p>Background</p> <p>Here we describe a new technical solution for optimization of <it>Pichia pastoris </it>shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in <it>P. pastoris </it>is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in <it>P. pastoris </it>is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.</p> <p>Results</p> <p>By applying on-line pO₂monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles.</p> <p>By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.</p> <p>Conclusion</p> <p>The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.</p

End-user-oriented telco mashups: The OMELETTE approach

With the success of Web 2.0 we are witnessing a growing number of services and APIs exposed by Telecom, IT and content providers. Targeting the Web community and, in particular, Web application developers, service providers expose capabilities of their infrastructures and applications in order to open new markets and to reach new customer groups. However, due to the complexity of the underlying technologies, the last step, i.e., the consumption and integration of the offered services, is a non-trivial and timeconsuming task that is still a prerogative of expert developers. Although many approaches to lower the entry barriers for end users exist, little success has been achieved so far. In this paper, we introduce the OMELETTE1 project and show how it addresses end-user-oriented telco mashup development. We present the goals of the project, describe its contributions, summarize current results, and describe current and future work. Copyright is held by the International World Wide Web Conference Committee (IW3C2)

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract

Bourdieusian Reflections on Language: Unavoidable Conditions of the Real Speech Situation

The main purpose of this paper is to shed light on Pierre Bourdieu’s conception of language. Although he has dedicated a significant part of his work to the study of language and even though his analysis of language has been extensively discussed in the literature, almost no attention has been paid to the factthat Bourdieu’s account of language is based on a number of ontological presuppositions, that is, on a set of universal assumptions about the very nature of language. This article aims to fill this gap in the literature by offering a detailed overview of 10 key features which, from a Bourdieusian point of view, can be regarded as inherent in language. On the basis of this enquiry,the study seeks todemonstrate that——contraryto commonbelief——there is not only a Bourdieusian sociology of language but also a Bourdieusian philosophy of language, which provides a useful theoretical framework for examining the unavoidable conditions of the real speech situation. The paper draws to a close by reflecting on the flaws and limitations of Bourdieu’s approach to language