<p>Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.</p>

<p>Mushrooms are believed to have a great potential for production of bioactive metabolites e. g. terpenes, a group of interesting compounds with diverse chemical properties such as antitumour and antibacterial activity. Cop6 is a terpene cyclase isolated from the mushroom <em>Coprinus cinereus</em> that catalyzes the cyclization of farnesyl diphosphate (FPP) to mainly α-cuprenene. In this study gas chromatography combined with mass spectroscopy (GC-MS) is used to analyze the product profile of Cop6 mutants created by PCR based site directed mutagenesis. The goal is to produce trichodiene, the parent hydrocarbon in the biosynthesis of trichothecene antibiotics and mycotoxins. Valine instead of tyrosine in amino acid position 195 resulted in cyclisation of (E)-β-Farnesene and (3Z,6E)-α-Farnesene besides the products of the wild type enzyme. Another mutant with aspartic acid instead of asparagine in position 224 resulted in the synthesis of β-Bisabolene except for α-cuprenene and methionine in position 74 instead of isoleucine killed the activity of the cyclase. Furthermore, an attempt to saturation of position 98 was made, resulting in four mutants. Two of them essentially killed the activity of the cyclase whereas two had minor effect of the product profile compared to the wild type. </p>

Worst-case analysis is often meaningless in practice. Some problems never reach the anticipated worst-case complexity. Other solutions get bogged down with impractical constants during implementation, despite having favorable asymptotic running times. In this thesis, we investigate these contrasts in the context of finding maximum flows in planar digraphs. We suggest analytic techniques that adapt to the problem instance, and present a structural property that concludes equivalence between shortest paths and maximum st-flow in planar graphs. The best known algorithm for maximum st-flow in directed planar graphs is an augmenting- paths algorithm with O(n) iterations. Using dynamic trees, each iteration can be implemented in O(log n) time. Long before, Itai and Shiloach showed that when s and t are on the boundary of a common face, the O(n)-iteration augmenting-paths algorithm is equivalent to Dijkstra's algorithm in the graph���s dual: the max st-planar st-flow problem can be solved with one single-source shortest-path computation. In this thesis we show that (a) when s and t are separated by p faces, the max st-flow can be found with at most 2p single-source shortest-path computations, which, using the linear-time shortest-paths algorithm for planar graphs, results in an O(np)-time algorithm, and (b) that the equivalence between augmenting-paths and Dijkstra's extends to the most general non-st-planar digraphs, using their half-infinite universal cover graph. / Graduation date: 2013

The purpose of this exploratory study was to examine and describe the learning projects of a selected sample of small business owners in a community in the Southeastern United States. The study included the revision and modernization of Tough’s (1971) Learning Project Interview Schedule. A total of 35 small business owners were interviewed using a modified version of Tough’s Learning Project Interview Schedule. The schedule consisted of 10 learning project and seven demographic items that were adapted or created by a collaborative research team at the University of Tennessee using Tough’s (1971) Interview Schedule. Data revealed that participants had a mean of 6.8 learning projects conducted over the previous 12-months. The learner was the primary planner of 55.9% of all learning projects with a mix of planners used in 22.7% of cases. This study found that African-Americans identified the learner as the primary planner in 71.9% of learning projects, higher than the overall mean. Demographic information revealed that a large majority (88.6%) of participants had at least an intermediate computer skill level. This was reflected in the use of technology for learning projects. The Internet was indicated as a resource in 43.3% of learning projects and was second only to print sources (54.2%). Technology played a key role in the learning projects of small business owners as it acted as both a primary source of information and as a secondary source for finding additional resources including content experts, print sources, and multimedia. Recommendations for further research include the need for additional studies on the preferences for, and impact of using technology for conducting learning projects. Specifically, research may explore the learner’s perception of benefits of various forms of technology for conducting learning projects.

The purpose of this study was to identify the key educational components of agritourism in Tennessee. The study strived to identify if knowledge of the agricultural industry increased due to agritourism experiences and if perception of the agriculture industry changed due to the experience. Additionally, the research sought to describe visitor demographics and characteristics.Following a pilot study in 2009, three agritourism venues throughout the state of Tennessee were used to collect surveys to create a pool of respondents and to identify their original knowledge of perceptions of the agriculture industry for the 2010 study. Additional data was gathered through an extensive post survey that gathered demographic information and respondents’ knowledge and perception levels based on several five point Likert scale questions. The responses were coded and entered by the primary investigator. The information was analyzed using means, standard deviations, and frequencies.The study revealed that respondents to agritourism venues do think that they experience are educational. The visitors expected an educational experience. Experiences at agritourism venues tend to increase consumer confidence slightly. Many respondents agree that they learn best through the hands on experience provided through the agritourism venues. Additionally, all respondents agreed that they would recommend the experience to others.

Im Rahmen der Arbeit werden hierarchisch strukturierte Silikakompositfasern präsentiert, deren Bildung ähnlich zu natürlichen Silifizierungsreaktionen verläuft. Als Analoga zu Proteinfilamenten in Silika Morphogeneseorganismen werden selbstorganisierte, funktionale Polyethylenoxid-Peptid-Nanobänder eingesetzt. Mit der Isolierung einheitlicher Nanokompositfasern wird gezeigt, dass die PEO-Peptid-Nanobänder eine starke Bindungsaffinität gegenüber Kieselsäure besitzen, diese aus sehr stark verdünnten Lösungen anreichern und deren Kondensation zu Silikanetzwerken kontrollieren können. In höheren Konzentrationen entstehen durch die peptidgeleitete Silifizierung der PEO-Peptid-Nanobänder spontan makroskopische Kompositfasern mit sechs Hierarchieebenen. Diese verbinden Längen von bis zu 3 cm und Durchmesser von 1-2 mm mit einer definierten Feinstruktur im Submikrometerbereich. Als Resultat der komplexen inneren Struktur und der Kontrolle der Grenzflächen zwischen Nanobändern und Silika wird eine Nanohärte erreicht, die schon ~1/3 der Härte von Bioglasfasern darstellt. Für die Elastizität (reduziertes Eindrückmodul) dagegen konnte durch den relativ hohen Anteil (~40%) an verformbaren, organischen Komponenten ein ~4-mal größer Wert im Vergleich mit Bioglasfasern bestimmt werden. Des Weiteren wird die Prozessierung der makroskopischen Kompositfasern in einem 2D-Plotprozess vorgestellt. Mit Verwendung der PEO-Peptid-Nanobänder als „Tinte“ können Kompositobjekte in beliebigen Formen geplottet werden, deren Linienbreite sowie anisotrope Ausrichtung der Nano- und Submikrometerstrukturelemente direkt mit der Plotgeschwindigkeit korrelieren. Außerdem können die Kompositobjekte als Vorstufen für orientierte, mesoporöse Silikaobjekte verwendet werden. Nachdem Calcinieren werden Silikastrukturen mit einer hohen spezifischen Oberfläche und in Plotrichtung ausgerichteten zylindrischen Poren erhalten. Im Kontrast zu den anorganisch-bioorganischen Kompositfasern sollten unter Ausnutzung ionischer Wechselwirkungen oder Metallkoordination Kompositmaterialien mit anderen mechanischen Eigenschaften dargestellt werden. Es wird gezeigt, dass durch Variationen in der Aminosäuresequenz des Peptidkerns, die Oberflächen der PEO-Peptid-Nanobänder gezielt mit funktionellen Gruppen versehen werden können. Eine gerichtete Vernetzung dieser modifizierten Nanobänder wurde nicht erreicht, dafür könnten die imidazolfunktionalisierten Nanobänder als eindimensionale Protonenleiter, die mit photochromen Gruppen (Spiropyran) funktionalisierten Nanobänder für die Modifizierung von Oberflächenpolaritäten oder für gerichtete Kristallisationsprozesse eingesetzt werden. / In this work hierarchical structured silica-composite fibers are presented, whose formation is similar to natural silicification processes. Self-assembled, functional poly(ethylene oxide) (PEO) peptide nanotapes were utilized as analogue to protein filaments in silicamorphogenese organism. Isolation of homogenous nano composite fibers demonstrates that the PEO peptide nanotapes have a high affinity to bind silicic acid. They are able to enrich silicic acid from very dilute solution and can control the silica condensation process. Macroscopic composite fibers spontaneously arise if the PEO peptide nanotapes in a higher concentration were mixed with the silica precursor. These exhibit six distinguishable levels of hierarchical order, spanning length scales from the nanometer up to millimeters in lateral and even centimeters in longitudinal dimensions. As a result of the inner structure, reinforced composite fibers were obtained, exhibiting 1/3 of the mechanical hardness of natural glass sponge spicules. The elasticity, which is considered as one limiting factor in optical glass fibers, could be enhanced 4-times due to the incorporation of an increased amount of polymer peptide nanotapes (~40%). In addition a 2D-plot process is introduced, in which the polymer peptide nanotapes acts as an ink. By injecting a solution of the nanotapes into a diluted silicic acid solution composite objects can be plotted in any desired way. The width of the plotted lines as well as the anisotropic orientation of the nano- and sub micrometer structure elements correlates directly to the plotting speed. Besides the composite objects can be utilized as precursors for oriented, mesoporous silica objects. After a calcination procedure silica structures with cylindrical pores, aligned in plot direction, and a high specific surface area were received. In contrast to the inorganic-organic composite fibers other composite materials with different mechanical properties should be created exploiting ionic interactions or metal coordination. A variation in the amino acid sequence of the peptide core leads to an aimed functionalisation of the nanotape surfaces. A directed networking of such nanotapes was not observed, but imidazole functionalised nanotapes could maybe be used as one dimensional proton conductors. The nanotapes, which were tagged with photo chromic spiropyran units, have the ability to be used for controlled crystallization processes or the modification of surface polarities.

Current knowledge about processes that generate long-distance dispersal of plants is still limited despite its importance for persistence of populations and colonization of new potential habitats. Today wild large mammals are presumed to be important vectors for long-distance transport of diaspores within and between European temperate forest patches, and in particular wild boars recently came into focus. Here we use a specific habit of wild boar, i.e. wallowing in mud and subsequent rubbing against trees, to evaluate epizoic dispersal of vascular plant diaspores. We present soil seed bank data from 27 rubbing trees versus 27 control trees from seven forest areas in Germany. The mean number of viable seeds and the plant species number were higher in soil samples near rubbing trees compared with control trees. Ten of the 20 most frequent species were more frequent, and many species exclusively appeared in the soil samples near rubbing trees. The large number of plant species and seeds – approximated > 1000 per tree – in the soils near rubbing trees is difficult to explain unless the majority were dispersed by wild boar. Hooked and bristly diaspores, i.e. those adapted to epizoochory, were more frequent, above that many species with unspecialised diaspores occurred exclusively near rubbing trees. Different to plant species closely tied to forest species which occur both in forest and open vegetation, and non-forest species were more frequent near rubbing trees compared with controls. These findings are consistent with previous studies on diaspore loads in the coats and hooves of shot wild boars. However, our method allows to identify the transport of diaspores from the open landscape into forest stands where they might especially emerge after disturbance, and a clustered distribution of epizoochorically dispersed seeds. Moreover, accumulation of seeds of wetness indicators near rubbing trees demonstrates directed dispersal of plant species inhabiting wet places between remote wallows. / Das aktuelle Wissen über Prozesse, die zur Fernausbreitung von Pflanzen führen, ist trotz ihrer Bedeutung für das Überleben von Populationen und die Besiedlung neuer potenzieller Habitate noch immer sehr begrenzt. Wildlebende Großsäuger sind heutzutage vermutlich wichtige Vektoren für den Ferntransport von Diasporen innerhalb und zwischen den einzelnen Waldflächen in Mitteleuropa, und speziell das Wildschwein (Sus scrofa L.) spielt dabei offenbar eine herausragende Rolle. Wir nutzen hier ein spezifisches Verhalten des Wildschweins – Suhlen im Schlamm und nachfolgendes Scheuern an sogenannten Malbäumen – um die epizoochore Ausbreitung von Gefäßpflanzen-Diasporen einzuschätzen. Dargestellt werden die Ergebnisse von Samenbank-Untersuchungen von 27 Malbäumen im Vergleich zu 27 Kontrollbäumen aus sieben Waldgebieten in Deutschland. Sowohl die mittlere Zahl lebensfähiger Samen als auch die Artenzahl waren höher in Bodenproben neben Malbäumen. Zehn der 20 in der Samenbank verbreitetsten Pflanzenarten hatten hier ihren Schwerpunkt, und viele Arten kamen ausschließlich in den neben Malbäumen gewonnenen Proben vor. Die große Zahl von Pflanzenarten und Samen – zumindest > 1000 pro Baum – im Boden an Malbäumen lässt sich nur durch die Aktivität der Wildschweine erklären. Mit Haken oder Borsten ausgestattete, d.h. an Epizoochorie angepasste Diasporen waren häufiger, aber auch viele Arten mit unspezialisierten Diasporen kamen ausschließlich in der Samenbank bei Malbäumen vor. Anders als weitgehend an Wald gebundene Pflanzenarten waren solche, die sowohl im Wald und im Offenland vorkommen, sowie nicht im Wald vorkommende Arten häufiger neben Malbäumen als neben Kontrollbäumen. Diese Befunde stimmen mit denen früherer Untersuchungen von Diasporenladungen im Fell und in den Hufen geschossener Wildschweine überein. Unsere Methode erlaubt darüber hinaus aber die Identifizierung des Diasporentransports aus dem Offenland in die Waldbestände, wo sie insbesondere nach Störungen keimen dürften, sowie einer ungleichmäßigen Verteilung epizoochor ausgebreiteter Diasporen. Außerdem zeigt die Akkumulation von Samen von Nässezeigern neben den Malbäumen eine gezielte Ausbreitung nasse Standorte bewohnender Pflanzenarten zwischen entfernt gelegenen Suhlen.

Enzymes are attractive catalysts in organic synthesis since they are efficient, selective and environmentally friendly. A large number of enzyme-catalyzed transformations have been described in the literature. If no natural enzyme can carry out a desirable reaction, one possibility is to modify an existing enzyme by protein engineering and thereby obtain a catalyst with the desired properties. In this thesis, the development of enantioselective enzymes and their use in synthetic applications is described.  In the first part of this thesis, enantioselective variants of Candida antarctica lipase A (CALA) towards α-substituted p-nitrophenyl esters were developed by directed evolution. A highly selective variant of CALA towards p-nitrophenyl 2-phenylpropanoate was developed by pairwise randomization of amino acid residues close to the active site. The E value of this variant was 276 compared to 3 for the wild type. An approach where nine residues were altered simultaneously was used to discover another highly enantioselective CALA variant (E = 100) towards an ibuprofen ester. The sterical demands of this substrate made it necessary to vary several residues at the same time in order to reach a variant with improved properties. In the second part of the thesis, a designed variant of Candida antarctica lipase B (CALB) was employed in kinetic resolution (KR) and dynamic kinetic resolution (DKR) of secondary alcohols. The designed CALB variant (W104A) accepts larger substrates compared to the wild type, and by the application of CALB W104A, the scope of these resolutions was extended. First, a DKR of phenylalkanols was developed using CALB W104A. An enzymatic resolution was combined with in situ racemization of the substrate, to yield the products in up to 97% ee. Secondly, the KR of diarylmethanols with CALB W104A was developed. By the use of diarylmethanols with two different aryl groups, highly enantioselective transformations were achieved. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows:<strong>  </strong>Paper 5: Submitted.

Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics. In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.

Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques. / <p>QC 20100726</p>