Plasmonische Substrate stellen ein leistungsstarkes Werkzeug für analytische Anwendungen dar. Neue plasmonische Substrate werden entwickelt, um das Spektrum ihrer Anwendungen und die Nachweisgrenzen der analytischen Spektroskopie zu erweitern. Diese Arbeit setzte sich zum Ziel, plasmonische Nanostrukturen mit Magnesiumfluorid zu beschichten. Magnesiumfluoridbeschichtungen sind zwar porös, weisen aber eine hohe mechanische Stabilität und außergewöhnliche optische Eigenschaften auf (niedrigen Brechungsindexes, großen optischen Fensters). Die Kombination dieser Eigenschaften mit den positiven Eigenschaften von plasmonischen Nanostrukturen kann zu fortschrittlichen plasmonischen Substraten für analytische Anwendungen führen. Diese Arbeit bietet zwei Ansätze für die Beschichtung der plasmonischen Nanostrukturen an die Core-Shell-Nanopartikelherstellung, die einen plasmonischen Core enthält und die Beschichtung von auf Glas immobilisierten plasmonischen Nanostrukturen. Über Metal@metal Fluoride Core-Shell-Nanopartikel wurde in der Literatur noch nichts berichtet. Daher Au@MgF2wurde ein Ansatz verfolgt, der auf dem Wissen über Metall-@Metalloxide und Metallfluoride@Metallfluoride basiert und die Synthese von Core-Shell-Nanopartikeln ermöglicht. Die erhaltenen Strukturen wurden mit elektronenmikroskopischen Methoden charakterisiert. Der zweite Ansatz bestand in der Immobilisierung von Goldnanopartikeln auf Glas und deren Beschichtung mit Magnesiumfluorid. Diese Fertigungsart verleiht eine hohe mechanische Stabilität und wissenswerte optische Eigenschaften an plasmonischen Substraten, die sich durch eine hohe nanoskopische Homogenität der Goldnanopartikelverteilung auszeichnen und optischer Signale, die echte analytische Anwendungen ermöglichen, ermittelt. Die Beschichtung von auf Glas mit Magnesiumfluorid immobilisierten Goldnanopartikeln führt zu einem sehr vielversprechenden Substrat , das in Zukunft für Sensorik und andere Anwendungen verwendet werden kann. / Plasmonic substrates can be a powerful tool for analytical applications. In order to broaden the spectrum of their applications and to push the detection limits of analytical spectroscopy, new plasmonic substrates are developed. The motivation of this work was to coat plasmonic nanostructures with magnesium fluoride. Coatings of magnesium fluoride are porous but exhibit high mechanical stability and extraordinary optical properties including a low refractive index and a wide optical window. Combining these properties with the beneficial properties of plasmonic nanostructures can lead to advanced plasmonic substrates for analytical applications. Two approaches for coating of the plasmonic nanostructures are proposed in this work: a core-shell nanoparticles fabrication and coating of plasmonic nanostructures immobilized on glass. The fabrication of Au@MgF2 core-shell nanoparticles turned out to be an extremely challenging approach. Such systems have not been reported in the literature yet. Therefore, an approach based on knowledge of metal@metal oxides and metal fluorides@metal fluorides core-shell nanoparticles synthesis was undertaken. The obtained structures were characterized using electron microscopy methods. Due to the numerous difficulties in the synthesis and characterization this way of coating plasmonic nanostructures with magnesium fluoride was not further processed. The approach based on immobilization of gold nanoparticles on glass and coating them with magnesium fluoride using a dip-coating method provides plasmonic substrates that are characterized by a high nanoscopic homogeneity of the gold nanoparticles distribution, a high mechanical stability, interesting optical properties and enhancement factors of optical signals that allow for real analytical applications. The coating of gold nanoparticles immobilized on the glass with magnesium fluoride results in very promising substrate that can be used for sensing and other applications in the future.

The current anthropogenic practices of generating single-use waste streams in agriculture, forestry and manufacturing industries have created a host of environmental health problems. Humankind's reliance on non-renewable resources for the production of food and materials, and its current approach to product design and development, are clearly unsustainable. One mitigation strategy to reducing industrial and municipal solid waste, as well as environmental pollution, can be found in using white rot fungi to valorize our planet's most abundant and regenerative natural resource – plant biomass containing lignocellulose. From residual dry plant matter, white rot fungi can be employed through a solid-state fermentation process to produce a variety of edible, nutrient-dense saprotrophic mushrooms in addition to biologically augmented composite materials. Under the framework of the circular economy, agricultural and forestry byproducts with fibers containing lignin, cellulose and hemicellulose may be used as a feedstock for the production of both food and biomaterials – keeping plant biomass revolving through multiple cycles of use and reuse for a variety of product outputs that are biodegradable and help to sequester carbon. In this study, mushrooms were grown on a variety of lignocellulosic substrates derived from agricultural and forestry residues. Hemp-based substrates performed the best of the feedstocks with regard to mushroom yield and mycelium colonization time. Additionally, a number of mycelium composite products were designed and fabricated in this study using residual lignocellulosic plant biomass, including: insulation bricks, acoustical panels, and biodegradable planter pots. In particular, spent mushroom substrate containing hemp hurd and other agricultural and forestry residues showed significant potential in upcycling lignocellulosic plant biomass for the production of both mushrooms and mycelium materials. Regenerative design practices demonstrated how food and materials can be generated from the same lignocellulosic feedstock; therefore, reducing waste, circulating products and materials, and ultimately regenerating nature. / Master of Science / Environmental pollution and natural resource scarcity have encouraged exploration into using biologically based materials for the production of more ecologically friendly products. By valorizing the Earth's most abundant, renewable natural resource for the production of food and materials– dry plant matter containing lignocellulose – waste is reduced, carbon is stored, and materials can remain upcycled through multiple generations of production. Lignocellulosic residues – natural fibers containing the biopolymers lignin, cellulose and hemicellulose – have recently been given increased attention due to their ability to be aggregated and grown into low-cost, lightweight materials using white rot fungi. Mushroom farming has historically valorized lignocellulosic agricultural and forestry residues to grow an edible, nutrient-dense food crop. This thesis investigates the potential of various agricultural and forestry residues for the production of mushrooms and mycelium-based lignocellulosic composites. Furthermore, this study explores the utilization of spent mushroom substrate for the production of several mycelium-based composite products within the framework of the circular economy. Hemp-based substrates demonstrated significant potential in both mushroom production and mycelium composite fabrication, outperforming other agricultural residues in this study with regard to mushroom yield and speed of mycelial growth of Pleurotus ostreatus. More research into the tunable lignocellulosic substrate compositions will continue to help advance mushroom production and mycelium-based composite generation as environmentally friendly materials and production practices continue to gain interest.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Cheryl Boyer / The nursery industry in the United States, particularly in the Great Plains region is growing; however, materials used in creation of artificial substrates used to grow ornamental nursery crops continue to increase in price. Eastern Redcedar (Juniperus virginiana L.) is an indigenous plant throughout much of the United States and, in the Great Plains, it has become a pest. Use of wood-based substrates (primarily composed of pine trees) has been proven effective in both nursery and greenhouse production. Eastern Redcedar chips (JVC) could become a local and sustainable resource for the horticulture industry throughout the Midwest. Experiments were conducted to determine if JVC could be used as a substrate to replace or supplement three major substrate components; pine bark (PB), perlite, or peat moss. Four studies evaluated ornamental crop growth: two focused on comparing nursery crop production in PB and JVC, one focused on greenhouse production in peat moss, and the last on plant propagation in perlite. The first experiment (Chapter 2) involved combining ratios of JVC and PB with two fertilizer rates to grow woody plants. It was shown that while higher levels of fertilizer produced larger plants, that plants grown at either rate of fertilizer showed the same growth trends. As JVC content increased more than 20%, growth measurements such as shoot dry weight and plant height decreased. This decrease in growth can be attributed to the physical properties of JVC, which showed that as JVC content increased so did airspace with a corresponding decrease in container capacity. A follow-up experiment (Chapter 4) evaluated several different particle sizes of JVC and a PB control. It showed that despite the different particle sizes JVC substrate produced less growth than plants grown in PB though plants grown in JVC were all similar to each other. Another experiment (Chapter 3) was conducted to evaluate if JVC as a replacement for peat moss in producing greenhouse-grown annual crops. JVC’s low container capacity hindered plant growth with each increase in JVC content associated with a decrease in growth. Finally a propagation experiment (Chapter 5) used a combination of finely-ground JVC and perlite to grow cuttings of woody and herbaceous plants. It was shown that, in most cases, JVC and perlite were equivocal rooting cuttings. This work demonstrates that JVC can be used as a PB and peat moss supplement, but not a replacement nursery and greenhouse crop production. Use of JVC as a perlite replacement for plant propagation is recommended.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / James K. Koelliker / This research provides a deterministic model of in-vessel composting, based on Monod’s growth kinetics, to mirror biological-mixture decomposition. Existing models predict temperature curves assuming a single temperature-range organism, using a soluble (simple sugar) substrate, with bacteria as the microorganism, and they ignore the different temperature range environments that impact the growth rates of mesophilic and thermophilic microorganisms. The new computer-simulated model, written in MATLAB® by The MathWorks, has six unique features. First, three major carbon chain substrate groups are utilized: soluble, hemicellulose/cellulose, and lignin. An additional substrate group is used for inert substrates. Second, three major microorganism groups are utilized: bacteria for soluble substrate, actinomycetes for cellulose substrate, and fungi for lignin substrate. Third, two temperature-range microorganisms are included: mesophilic and thermophilic. Fourth, the model accounts for the death of microorganisms as the temperature transitions between the temperature ranges. Most of the dead cellular mass is returned to soluble substrate for reutilization and a portion is considered resistant to biological decomposition and is added into the lignin substrate. Fifth, stoichiometric equations account for substrate and microorganism compositions, oxygen and nitrogen requirements, and carbon dioxide and water production. Sixth, the relationship between biological activity and water is better defined. Experimental research was conducted to validate the model. Laboratory analysis distinguished the substrate types. The results indicate the model did differentiate between different levels of substrate types, and the mesophilic and thermophilic microorganism types. Also, the model did differentiate between the bacteria, actinomycetes and fungi. The influence was small, however, because of the different maximum growth rates of the three types of microorganisms. Returning dead microbes to the substrate pools as a result of temperature transitions affected the model results positively. Additional research is needed to account for the influence of volume reduction, develop a better microbial growth curve, include particle size influence, add temporal temperature fluctuations to the external boundary conditions, incorporate pH and nitrogen availability, and develop a three-dimensional model. KEY WORDS. Aerobic composting, mathematical composting model, substrate types, microorganism types, microorganism temperature range, mesophilic, thermophilic, microbial death utilization, moisture composting relationship.

Focusing on reducing the air consumption of an air flotation rail system, a flowrate-amplified flotation element was recently developed. This new flotation element ulitises the rotational flow to intake extra air via an intake hole, and thus, effectively improves the flotation height. Compared to a conventional flotation element, the flowrate-amplified flotation element can reduce air consumption by approximately 50% for the same load and flotation height. To gain an understanding of the flow phenomenon in the flowrate-amplified flotation element, experiments and CFD simulations are conducted in this study. Based on the results, we found that the flowrate-amplified flotation element could take a part of the kinetic energy of the rotating air to suck in extra air. The intake hole greatly affects the pressure field and velocity field of the flotation element. Additionally, the effects of the variant gap height and supplied flow rate were also discussed. The results indicate that the pressure distribution decreases as the gap height increases and increases as the supplied flow rate increases.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects one third of the world’s population and kills 1.7 million people per year. The increasing prevalence of multi- and extensively drug resistant M. tuberculosis strains means that there is an urgent need to develop new anti-TB drugs. The genome of M. tuberculosis has five copies of the ESAT-6 gene clusters (ESX-1, -2, -3, -4 and -5), which are essential for the survival (ESX-3) and pathogenicity (ESX-1 and ESX-5) of the bacterium. The ESX clusters encode for proteins which form a novel secretion system which has been shown to secreted small T-cell antigens of the esx gene family, as well as other proteins such as the PE and PPE’s. The mycosins are a family of genes situated in the ESX clusters which encode for putative subtilisin-like serine proteases. These proteins are the most conserved proteins within the five clusters. Apart from their conserved protein sequence, mycosin-3 is also an essential protein specific to the mycobacteria, which makes it an attractive potential drug target. Identifying the substrate(s) of mycosin-3 could help to understand the function of this enzyme and discover novel inhibitors from which new drugs could be designed. We hypothesize that the secreted products of the ESX system could be potential substrates for the mycosins. Specifically, we hypothesize that PE5, PPE4, esxG and esxH (all found in ESX-3) might be the substrates for mycosin-3. Mycosin-3, PE5, PPE4, esxG and esxH were thus cloned, expressed and purified respectively. The four substrates were used for protease assays using mycosin-3 as the protease. The protease-substrate mixture were subsequently separated on 2-D SDS-PAGE gels to check whether there were any cleavage of the four substrates. Although all the target fusion proteins were cloned and expressed successfully, the protease assay results showed no cleavage for any of the four substrates. Possible explanations for the failure of cleavage were: (1) impure enzyme and substrate(s); (2) inappropriate buffer conditions; (3) the hypothesized substrates might not be the substrates of mycosin-3; and (4) incorrect folding or modification of the target fusion proteins might have taken place. Future research will aim to address these possible limitations in order to fully elucidate the function and substrate specificity of mycosin-3 and to use this information for the design of novel drugs against M. tuberculosis. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat tuberkulose (TB) veroorsaak, infekteer `n derde van die wêreld se bevolking en veroorsaak die dood van 1.7 miljoen mense per jaar. Die verhoogde voorkoms van multi- en ekstensiewe middelweerstandige stamme van M. tuberculosis beteken dat daar `n ernstige nodigheid is om nuwe anti-TB middels te ontwikkel. Die genoom van M. tuberculosis het vyf kopieë van die ESAT-6 geengroepe (ESX-1, -2, -3, -4 en -5), wat essensieel is vir die oorlewing (ESX-3) en patogenisiteit (ESX-1 and ESX-5) van die bakterium. Die ESX groepe enkodeer vir proteïene wat `n nuwe uitskeidingssisteem vorm wat bewys is om klein T-sel antigene van die esx geenfamilie, sowel as ander proteïene soos die PE en PPE proteïene uit te skei. Die mycosins is `n familie gene wat in die ESX geengroepe voorkom en wat waarskynlik enkodeer vir subtilisin-agtige serine proteases. Hierdie proteïene is die mees gekonserveerde proteïene in die vyf geengroepe. Mycosin-3 is `n essensiële protein wat spesifiek in die mikobakteriëe voorkom, sodat dit `n aantreklike teiken vir die ontwikkeling van middels is. Die identifisering van die substrate van mycosin-3 kan moontlik help om die funksie van die ensiem te verstaan en om nuwe inhibeerders vir die ensiem te ontdek, wat kan lei tot die onwikkeling van nuwe middels. Ons hipotese is dat die uitgeskeide proteïene van die ESX sisteem moontlik die substrate van die mycosin proteïene kan wees. Meer spesifiek, ons hipnotiseer dat die proteïene PE5, PPE4, esxG en esxH (wat almal in ESX-3 voorkom) die substrate vir mycosin-3 kan wees. Mycosin-3, PE5, PPE4, esxG en esxH is afsonderlik gekloneer, uitgedruk en gesuiwer. Die vier substrate is gebruik vir protease proewe met mycosin-3 as die protease. Die protease-substraat mengsel is hierna deur middel van 2-D SDS-PAGE geanaliseer om te kyk of daar enige kliewing van die vier substrate voorgekom het. Alhoewel al die teiken fusieproteïene suksesvol gekloneer, uitgedruk en gesuiwer is, het die protease proewe geen kliewing getoon vir enige van die vier potensiële substrate nie. Moontlike verklarings vir hierdie negatiewe resultaat is die volgende: (1) ensiem en substrate was moontlik onsuiwer; (2) bufferkondisies was moontlik nie korrek nie; (3) gehipotiseerde substrate mag moontlik nie substrate van mycosin-3 wees nie; en (4) nie-korrekte vouing of modifisering van die teiken proteïene kon moontlik plaasgevind het. Toekomstige navorsing sal daarop gemik wees om hierdie beperkinge aan te spreek om sodoende die funksie en substrate van mycosin-3 te kan ontdek en nuwe middels teen M. tuberculosis te ontwerp.

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes is how they are able to recognize a variety of seemingly unrelated amino acid sequences as cleavage sites. We are investigating the basis for this general substrate recognition in neuropeptidases using thimet oligopeptidase (TOP) as a model. Crystal structures of TOP in complex with a variety of substrates and inhibitors shed light on the mechanisms underlying substrate recognition and pave the way for re-targeting substrate recognition in these enzymes. Nano test tube particles have been proposed as a means of delivering therapeutics such as enzymes. However, the template synthesis method for nano test tube production does not produce therapeutic quantities. In order to take full advantage of re-engineered neuropeptidases a new method for nano test tube synthesis has been developed. We show that a non-destructive template synthesis methodology can be applied to produce nano test tube particles in quantities useful for therapeutic enzyme immobilization.

Cell cultures provide researchers the opportunity to observe cell behavior in response to specific, well-defined environmental cues, leading to insights that enable better engineering design for tissue culture and other biomedical applications. Chemical and electrical stimuli have been successfully applied to cultured cells to approximate aspects of the dynamic conditions experienced in vivo. However, in vitro topographical cues have mostly been limited to static substrates that do not subject cells to the dynamic conditions they experience in vivo when tissue remodels during development and wound healing. Delivering dynamic topographical cues to cultured cells can answer long-standing questions about mechanisms of cell morphology changes. Such capabilities could also facilitate engineering of wound-healing matrices and nerve guidance conduits by promoting migration of cells and providing directional guidance to cellular processes. This dissertation describes the development of approaches for introducing in situ topographical cues to cell cultures and inducing responses such as neurite guidance and cell alignment. Both strategies undertaken in this work make use of multiphoton-promoted photochemistry to print and manipulate three-dimensional microscopic protein hydrogel structures. In one approach, a technique referred to as micro-3D printing, topographical guidance cues are printed in the proximity of cultured cells to guide the growth of cellular processes. By translating a tightly-focused pulsed laser beam through a printing reagent solution flooding cultured cells, features are printed that provide physical guidance to extending neurites from NG108-15 cells, a neuronal model cell type. In another approach, an innovative technique known as micro-3D imprinting is developed for producing micrometer-scale depressions on the surfaces of photoresponsive protein hydrogels. The impact of various experimental parameters on topographical feature dimensions is characterized. Micro-3D imprinting is used to introduce dynamic topographical changes on a cell culture substrate, demonstrating that NIH-3T3 cells, a fibroblast cell model, alter their morphology and alignment in response to the introduction of a grooved surface topography. This set of approaches introduces new tools to the repertoire of cell biologists for exploring the behavior of cells growing in a spatio-temporally dynamic environment, opening possibilities for studies of cellular behavior in conditions that may better reflect environments cells experience in vivo. / text

<p>Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates. </p><p>Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates. </p><p>Several multivariate analysis tools, <i>e.g.</i> principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.</p>

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.