NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIO-OLIGOMERS

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications. To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The dissertation will present the details about the development of the techniques. Chapter 1 will make an introduction to oligodeoxynucleotides (ODNs), their synthesis and purification. Chapter 2 will describe the detailed studies of using the catching failure sequences by polymerization method to purify ODNs. Chapter 3 will describe the further optimization of the catching failure sequences by polymerization ODN purification technology to the level of practical use. Chapter 4 will present using the catching full-length sequence by polymerization method for ODN purification using acid-cleavable linker. Chapter 5 will make an introduction to peptides, their synthesis and purification. Chapter 6 will describe the studies using the catching full-length sequence by polymerization method for peptide purification.

THE FANNED FLAMES OF DISCONTENT: A SOLIDARITY-INSPIRED HISTORY OF THE IDENTITY/IDEOLOGY, CULTURAL HISTORY, AND RHETORICAL STRATEGIES OF THE WOBBLIES DURING THE 1916 MINNESOTA IRON ORE STRIKE

Rooted in critical scholarship this dissertation is an interdisciplinary study, which contends that having a history is a basic human right. Advocating a newly conceived and termed, Solidarity-inspired History framework/practice perspective, the dissertation argues for and then delivers a restorative voice to working-class historical actors during the 1916 Minnesota Iron Ore Strike. Utilizing an interdisciplinary methodological framework the dissertation combines research methods from the Humanities and the Social Sciences to form a working-class history that is a corrective to standardized studies of labor in the late 19th and early 20th centuries. Oftentimes class interests and power relationships determine the dominant perspectives or voices established in history and disregard people and organizations that run counter to, or in the face of, customary or traditional American themes of patriotism, the Protestant work ethic, adherence to capitalist dogma, or United States exceptionalism. This dissertation counteracts these traditional narratives with a unique, perhaps even revolutionary, examination of the 1916 Minnesota Iron Ore Strike. The intention of this dissertation's critical perspective is to poke, prod, and prompt academics, historians, and the general public to rethink, and then think again, about the place of those who have been dislocated from or altogether forgotten, misplaced, or underrepresented in the historical record. Thus, the purpose of the dissertation is to give voice to historical actors in the dismembered past. Historical actors who have run counter to traditional American narratives often have their body of "evidence" disjointed or completely dislocated from the story of our nation. This type of disremembering creates an artificial recollection of our collective past, which de-articulates past struggles from contemporary groups seeking solidarity and social justice in the present. Class-conscious actors, immigrants, women, the GLBTQ community, and people of color have the right to be remembered on their own terms using primary sources and resources they produced. Therefore, similar to the Wobblies industrial union and its rank-and-file, this dissertation seeks to fan the flames of discontented historical memory by offering a working-class perspective of the 1916 Strike that seeks to interpret the actions, events, people, and places of the strike anew, thus restoring the voices of these marginalized historical actors.

E-mail-“inspired” changes in non-native legal discourse

At IDC, students use electronic resources for research and online interactive communication with instructors, usually in English. This paper discusses preliminary research into the overlap between the informality of e-mail communication between students and instructors and the growing use (or misuse) of e-mail-type informal discourse in formal written legal assignments. Four students were given a hypothetical legal case and requested to write: (a) a formal letter that would be sent by e-mail to one of the parties in the case, and (b) an executive memo e-mail to the senior partner in one of the law firms representing the parties. No instruction was given as to constructing a formal legal letter or an executive memo. In the resulting e-mail communications, many examples of typical informal e-mail shorthand were used. The students were interviewed and were able to locate and change most of the errors in their letters. Several students expressed the belief that this type of “shorthand” is or should be acceptable when the formal message is an e-mail communication.

Why Do We Think Practice Research is a Good Idea? Comments and Musings Inspired by the Salisbury Statement

In this paper I raise some questions about current understandings of practice research and whether they are worth pursuing. In particular, the notion of a gap between practice and research is examined in terms of how it constricts thinking about this issue. I also attempt to explicate some of the less examined assumptions associated with practice research. Finally, I suggest that we embrace multiplicity, not by trying to accommodate all views under the practice research umbrella, but by accepting that there will be many versions of practice research that will have differential appeal.

Generierung von Konstruktionsmerkmalen zur Entwicklung eines bio-inspirierten Kommissionier- Greifers für die Intralogistik mittels Clusteranalyse

Kurzbeschreibung: In der Automatisierung von intralogistischen Kommissioniervorgängen ist ein großes Zukunftspotential erkennbar. Elementarer Bestandteil des Automatisierungsprozesses ist der Einsatz von Industrierobotern, die mit einem geeigneten Endeffektor, dem Greifer, ausgestattet werden müssen. Die Robotik ist in der Lage schneller, präziser und ausdauernder als die menschlichen Kommissionierer zu arbeiten und trägt damit entscheidend zur Effizienzsteigerung bei. Eine wesentliche Herausforderung für diesen Entwicklungsschritt zur Substitution der manuellen Kommissionierung ist die Konstruktion und Bereitstellung eines geeigneten Greifsystems. Am Lehrstuhl für Maschinenelemente und Technische Logistik der Helmut-Schmidt-Universität wurde mit der Erfahrung aus einem vorangegangenen Forschungsprojekt die Methode der Clusteranalyse erstmalig zur Untersuchung von Greifobjekten zur Entwicklung eines bionischen Universalgreifers für die Kommissionierung von Drogerieartikeln verwendet. Diese Abhandlung beschreibt einen Beitrag zur Entwicklung dieses Greifers am Beispiel handelsüblicher Drogerieartikel, die aktuell manuell kommissioniert werden. Diese werden hinsichtlich der für das Greifen relevanten Objektmerkmale geclustert und die daraus resultierenden Erkenntnisse in Form von Konstruktionsmerkmalen abgeleitet. Nach einer Analyse und Festlegung der greifrelevanten Merkmale der Greifobjekte wird eine Objektdatenbasis erstellt. Mit Hilfe geeigneter Methoden wird die gewonnene Datenbasis aufbereitet und reduziert. Im Anschluss werden die Greifobjekte bzw. deren Merkmalsausprägungen einer hierarchischen Clusteranalyse unterzogen. Hierbei werden die Grenzen der gebildeten Cluster mittels der zugehörigen Greifobjekte festgelegt und analysiert. Abschließend werden bestimmte greiferspezifische Merkmale auf die Anwendbarkeit in den Clustern überprüft und bewertet. Diese Betrachtungen ermöglichen es, dass spezielle Anforderungen an den Greifer, die direkt aus den Eigenschaften der Greifobjekte herrühren, zuverlässig erkannt und konstruktiv berücksichtigt werden können.

Carbon dioxide and climate impulse response functions for the computation of greenhouse gas metrics: a multi-model analysis

The responses of carbon dioxide (CO2) and other climate variables to an emission pulse of CO2 into the atmosphere are often used to compute the Global Warming Potential (GWP) and Global Temperature change Potential (GTP), to characterize the response timescales of Earth System models, and to build reduced-form models. In this carbon cycle-climate model intercomparison project, which spans the full model hierarchy, we quantify responses to emission pulses of different magnitudes injected under different conditions. The CO2 response shows the known rapid decline in the first few decades followed by a millennium-scale tail. For a 100 Gt-C emission pulse added to a constant CO2 concentration of 389 ppm, 25 ± 9% is still found in the atmosphere after 1000 yr; the ocean has absorbed 59 ± 12% and the land the remainder (16 ± 14%). The response in global mean surface air temperature is an increase by 0.20 ± 0.12 °C within the first twenty years; thereafter and until year 1000, temperature decreases only slightly, whereas ocean heat content and sea level continue to rise. Our best estimate for the Absolute Global Warming Potential, given by the time-integrated response in CO2 at year 100 multiplied by its radiative efficiency, is 92.5 × 10−15 yr W m−2 per kg-CO2. This value very likely (5 to 95% confidence) lies within the range of (68 to 117) × 10−15 yr W m−2 per kg-CO2. Estimates for time-integrated response in CO2 published in the IPCC First, Second, and Fourth Assessment and our multi-model best estimate all agree within 15% during the first 100 yr. The integrated CO2 response, normalized by the pulse size, is lower for pre-industrial conditions, compared to present day, and lower for smaller pulses than larger pulses. In contrast, the response in temperature, sea level and ocean heat content is less sensitive to these choices. Although, choices in pulse size, background concentration, and model lead to uncertainties, the most important and subjective choice to determine AGWP of CO2 and GWP is the time horizon.

Nano-mechanical transduction of polymer micro-cantilevers to detect bio-molecular interactions

Using variothermal polymer micro-injection molding, disposable arrays of eight polymer micro-cantilevers each 500 μm long, 100 μm wide and 25 μm thick were fabricated. The present study took advantage of an easy flow grade polypropylene. After gold coating for optical read-out and asymmetrical sensitization, the arrays were introduced into the Cantisens(®) Research system to perform mechanical and functional testing. We demonstrate that polypropylene cantilevers can be used as biosensors for medical purposes in the same manner as the established silicon ones to detect single-stranded DNA sequences and metal ions in real-time. A differential signal of 7 nm was detected for the hybridization of 1 μM complementary DNA sequences. For 100 nM copper ions the differential signal was found to be (36 ± 5) nm. Nano-mechanical sensing of medically relevant, nanometer-size species is essential for fast and efficient diagnosis.

Comparison of the toxicity of diesel exhaust produced by bio- and fossil diesel combustion in human lung cells in vitro

Alternative fuels are increasingly combusted in diesel- and gasoline engines and the contribution of such exhausts to the overall air pollution is on the rise. Recent findings on the possible adverse effects of biodiesel exhaust are contradictive, at least partly resulting from the various fuel qualities, engine types and different operation conditions that were tested. However, most of the studies are biased by undesired interactions between the exhaust samples and biological culture media. We here report how complete, freshly produced exhausts from fossil diesel (B0), from a blend of 20% rapeseed-methyl ester (RME) and 80% fossil diesel (B20) and from pure rapeseed methyl ester (B100) affect a complex 3D cellular model of the human airway epithelium in vitro by exposing the cells at the air–liquid interface. The induction of pro-apoptotic and necrotic cell death, cellular morphology, oxidative stress, and pro-inflammatory responses were assessed. Compared to B0 exhaust, B20 exhaust decreased oxidative stress and pro-inflammatory responses, whereas B100 exhaust, depending on exposure duration, decreased oxidative stress but increased pro-inflammatory responses. The effects are only very weak and given the compared to fossil diesel higher ecological sustainability of biodiesel, it appears that – at least RME – can be considered a valuable alternative to pure fossil diesel.