Datura metel is a herbaceous plant found in almost all tropical parts of the world. It belongs to the family Solanaceae whose members, viz. Duboisia, Atropa, Hyoscyamus and Datura plants are known to produce tropane alkaloids- hyoscyamine and scopolamine which are most noted for their therapeutic use as anti-cholinergic agents. Since these alkaloids are produced in very low amounts in plants, alternative sources and methods of production for these alkaloids have been crucial in meeting the demands for these drugs. Endophytic fungi inhabiting a plant may have the potential to produce the same compounds as the host plants. The aim of the present study was to search for tropane alkaloid producing endophytic fungal isolates from Datura metel. Eighteen endophytic fungi were isolated from various tissues of Datura metel and screened for the presence of three tropane alkaloid biosynthetic genes- putrescine N-methyltransferase (PMT), tropinone reductase I (TRI) and hyoscyamine 6β-hydroxylase (H6H) using PCR-based screening approach. Six endophytic fungal isolates were found to possess the PMT, TR1 and H6H genes. The fungi were identified using molecular taxonomy as Col letotrichum boninense, Phomopsis sp., Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col letotrichum gloeosporioides and the identity was confirmed using colony and spore morphology. The production of tropane alkaloids hyoscyamine and scopolamine by the fungi has been ascertained using various techniques like TLC, HPLC and ESI-MS/MS by comparison with the authentic reference standards. The amount of tropane alkaloids produced by all six fungi in liquid cultures was quantified using HPLC analysis. Among the six tropane alkaloid-producing fungi Col letotrichum incarnatum gave the highest yields of hyoscyamine and scopolamine which were 3.906 mg/L and 4.13 mg/L, respectively. With an aim to characterize the tropane alkaloid biosynthetic genes in these fungi, the PMT gene was isolated from five of the endophytic fungi- Col letotrichum boni-nense, Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col-letotrichum gloeosporioides for the first time and the sequence analysis showed high ho-mology (98%) to the Datura metel PMT cDNA. The gene was found to be devoid of introns in the fungi. Further phylogenetic analysis of the full length PMT sequence from the fungi strongly supports the hypothesis of horizontal gene transfer between the host plant and endophytic fungi. For further in detail characterization of fungal PMT, the Col letotrichum boninense PMT gene was taken as a representative. CbPMT gene was cloned in pRSET A expres-sion vector and heterologously expressed in E. coli and biochemically characterized. For optimal yield of soluble protein upon heterologous expression different conditions such as IPTG concentration, temperature and time post induction were optimized. Optimal yield was obtained by inducing the culture by 0.25 mM IPTG once it had reached and O.D. of 0.6 and incubating at 37◦ C for 3 h. The recombinant CbPMT enzyme expressed as histidine tagged fusion protein was purified using Ni-NTA affinity chromatography. Gel elution studies were carried out to determine molecular weight of the protein and it was found that the protein exists as a homodimer in solution with some amount also present as a monomer. Catalytic activity of the purified recombinant enzyme was studied for its dependence on both substrates putrescine as well as S-adenosylmethionine (SAM). The Km and Vmax values for putrescine were found to be 464 µM and 18.55 nkat/mg, respectively, while those for S-adenosylmethionine were found to be 628 µM and 18.63 nkat/mg, respectively. Optimum temperature for activity was found to be 37◦ C and optimum pH range was found to be 8-9. Fluorescence spectroscopy was used to study the binding affinity of both the sub-strates to the enzyme. Fluorescence quenching data for each substrate was analysed by using a nonlinear regression curve fit and Kd values were found to be 0.309 mM for pu-trescine and 0.118 mM for SAM, respectively. Circular dichroism spectrum of the enzyme indicated a pattern typical for alpha helix in the secondary structure. Binding of either substrate led to increase in ellipticity of the protein. Fluorescence quenching studies with collisional quenchers- acrylamide, potassium iodide, and cesium chloride indicated that the native protein is folded in a conformation that allows tryptophan residues to be acces-sible for quenching. The fraction of tryptophan residues (fa ) accessible for quenching by acrylamide (1.06) was found to be higher than that for potassium iodide (0.54) while that cesium ions was the least (0.38). The neutral quencher acrylamide could access all the tryptophans meaning that none of tryptophans are completely buried inside hydrophobic cores. the differential accessibility to the charged quenchers, however, indicates that more of the tryptophans are surrounded by positively charged amino acids. The unfolding of the protein was studied with the aid of chaotropic agents guanidine-HCl and urea and thermodynamic parameters were determined. The denaturant m-values were found to be 2.313 kcal/mol/M for Gdn-HCl and 2.345 kcal/mol/M for urea respectively. The free energy of unfolding was estimated to be 2.635 kcal/mol for Gdn-HCl and 4.630 kcal/mol for urea. Since no reports are available about the thermodynamics of folding and unfolding of PMT from any plant source, this study contributes towards the understanding of protein stability. Although a lot of reports are available on the biochemical characterization of PMT from different plant sources, the crystal structure of PMT is not yet available. In the current work, homology based modelling studies on CbPMT were carried out to get some idea about the protein tertiary structure. Homology based modelling studies showed that a significant amount of protein is present as α-helices which are present on the surface while the β-sheets are present in the interior of the protein. Each monomer of the protein is capable of binding both the substrates and hence the dimerization property of the enzyme could be a purely structural one leading to more stability and solubility of the protein. In conclusion, this study has shown for the first time that endophytic fungi have significant potential to be used for tropane alkaloid production and six such fungal strains have been identified. Although the production of tropane alkaloids by endophytic fungi is not very high, it can be scaled up by over-expressing the biosynthetic gene putrescine N-methyltransferase in the highest producer- Col letotrichum incarnatum to further increase the yield. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for these drugs economically.

This thesis presents new models for determining piezoresistive response in long, thin polysilicon beams with either axial and bending moment inducing loads or torsional loads. Microelectromechanical (MEMS) test devices and calibration methods for finding the piezoresistive coefficients are also presented for both loading conditions. For axial and bending moment inducing loads, if the piezoresistive coefficients are known, the Improved Piezoresistive Flexure Model (IPFM) is used to find the new resistance of a beam under stress. The IPFM first discretizes the beam into small volumes represented by resistors. The stress that each of these volumes experiences is calculated, and the stress is used to change the resistance of the representative resistors according to a second-order piezoresistive equation. Once the resistance change in each resistor is calculated, they are combined in parallel and series to find the resistance change of the entire beam. If the piezoresitive coefficients are not initially known, data are first collected from a test device. Piezoresistive coefficients need to be estimated and the IPFM is run for the test device's different stress states giving resistance predictions. Optimization is done until changing the piezoresistive coefficients provides model predictions that accurately match experimental data. These piezoresistive coefficients can then be used to design and optimize other piezoresistive devices. A sensor is optimized using this method and is found to increase voltage response by an estimated 10 times. For torsional loads, the test device consists of a slider-crank connected to two torsional legs. The slider-crank creates torsional stress in the legs which causes a change in the electrical resistance through the legs. A model that predicts the effects of a scissor hinge on the slider-crank is presented. Torsional stresses in the legs are calculated delete{using the membrane analogy.} and the legs are discretized into long parallel resistors and the stresses delete{from the membrane analogy} applied to each resistor. Assuming a second-order piezoresistance, an optimization is then done to find the piezoresistive coefficients by changing them until the model prediction fits the test data. These coefficients can be used to predict angular displacement from resistance measurements in fully integrated torsional sensors. Potential applications are discussed, and a torsional accelerometer is presented.

Das IceCube Neutrino-Observatorium ist ein am geografischen Südpol im Eis installierter Neutrinodetektor. In IceCube werden Neutrinos mit Tscherenkow-Strahlung von Sekundärteilchen aus Neutrino Interaktionen detektiert. Für den Nachfolgedetektor IceCube-Gen2, werden neue und verbesserte Lichtdetektoren gesucht. Die vorliegende Arbeit beschreibt die Entwicklung eines dieser Lichtdetektoren. Dieser basiert auf Wellenlängen schiebenden und Licht leitenden Technologien. Der Detektor mit dem Namen "Wavelength-shifting Optical Module" (WOM) verwendet eine transparente Röhre, mit wellenlängenschiebender Farbe, als passiver Photonendetektor. Das in der Wellenlänge verschobene Licht wird durch Totalreflexion, zu kleinen PMTs an beiden Enden geleitet. Die Auswahl dieses Designs reduziert die Kosten und verbessert das Signal-Rausch-Verhältnis wesentlich, möglicherweise können mit dieser Lösung extragalaktische Supernova in zukünftigen Detektoren beobachtet werden. Als eine Kernkomponente wird die wellenlängenschiebende Röhre ausführlich untersucht. Verschiedene Messaufbauten und Auswertungsmethoden werden entwickelt, um diese im Anschluss zu untersuchen und zu bewerten. Iterative Verbesserungen der Materialien und des Farbauftrageverfahren als auch Messmethoden, resultieren in einer kombinierten Einfang-, Wellenlängenschiebe- und Transporteffizienz von 28,1 +/- 5,4 % der Röhre. Ein Model zur Beschreibung des Lichtverhaltens in der Röhre wird entwickelt um eine Diskrepanz zwischen Theorie und Messung zu untersuchen. Die Kombination zwischen Messung und Model, bestätigt die Aussagekraft des Models und zeigt, dass ein Großteil der Verluste beim Lichttransport zustande kommen. Darüber hinaus werden die physikalischen Eigenschaften des WOM in die IceCube Simulationsumgebung eingebaut. Der Vergleich zu einem Konkurrenzmodul zeigt eine Überlegenheit des WOM um den Faktor 1,05 +/- 0,07. Es werden Vorschläge und Ausblicke für Verbesserungen der Leistungsfähigkeit des WOMs gegeben. / The IceCube Neutrino Observatory is an in ice neutrino detector located at the geographic South Pole. In IceCube neutrinos are detected via Cherenkov light produced by secondary particles in neutrino interactions. For the upgraded detector IceCube-Gen2, new and improved light detectors are sought-after. This work describes the development of one of those light detectors based on a novel combination of wavelength-shifting and light-guiding technology. The detector named the Wavelength-shifting Optical Module (WOM) utilizes a large transparent tube, coated with wavelength-shifting paint as a passive photon detector. The wavelength-shifted light is guided via total internal reflection towards small active light detectors, at each end of the tube. This design reduces costs and improves the signal to noise ratio significantly, thereby potentially enabling extragalactic supernova detections in future detectors. As a core component, the wavelength-shifting tube is extensively investigated. Different measurement setups and evaluation techniques are developed and investigated. Iterative improvement of materials and coating techniques as well as measurement methods currently result in a combined photon capture, shift and transport efficiency of 28.1 +/- 5.4 % for the tube. Those results contrast the theoretical maximum of 74.5 %. A model is developed to describe the light propagation and loss processes in the tube and to understand the discrepancies between theory and measurement. The combination of the measurements with the model, validate the descriptive qualities of the model and show that most of the light is lost during the light propagation in the tube. Additionally, the physical properties of the WOM are included in the IceCube simulation framework. A comparison to a competing module showed that the WOM outperforms by a factor of 1.05 +/- 0.07 in photon detection numbers. Where applicable, suggestions and outlooks are given to enhance the performance of the WOM.

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