Mainz, Univ., Diss., 2001.

Organophosphorus (OP) pesticide toxicity is a global health problem. Respiratory failure due to neuromuscular transmission dysfunction accounts for about 300,000 deaths annually in rural Asia. However, the clinical manifestation is complex, and described in terms of acute, intermediate, and chronic syndromes. The underlying mechanism of toxicity is still unclear. OP pesticides contain inhibitors of acetylcholinesterase (AChE), for example dimethoate, emulsified in an organic solvent, typically cyclohexanone. A hypothesized mechanism is initial excitotoxicity through inhibition of acetylcholinesterase followed by failure of neuromuscular synaptic transmission. I tested this electrophysiologically in vitro by measuring properties of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in isolated sciatic nerve/flexor digitorum brevis muscles from mice, bathed in HEPES-buffered mammalian physiological saline (MPS). Muscle action potentials were abolished with μ-conotoxin (2μM). First, we tested the effects of plasma taken from Göttingen minipigs instilled orally (isofluorane anaesthesia) with a formulated pesticide (2.5ml/kg) whose active ingredient is dimethoate dissolved in cyclohexanone. This plasma abolished evoked synaptic transmission and increased spontaneous MEPP frequency within 60-180 minutes of bath application. However plasma from minipigs instilled with dimethoate alone produced no failure of transmission. Plasma contained either pesticide or dimethoate significantly increased the half decay time of EPPs. However, pesticide-plasma also contained the metabolites omethoate (100μM) and cyclohexanol (5 mM). We found that bath application of omethoate alone caused a potent dose-dependent increase in EPP decay time. Cyclohexanol (5 mM) also increased EPP decay time but it also decreased both the excitability of axons and MEPP amplitude. In combination, omethoate and cyclohexanol produced greater disruption of neuromuscular transmission than either dimethoate or cyclohexanone, alone or in combination and this was particularly evident in isometric tension recordings, in which prolonged after-contraction and slow relaxation were observed during and immediately following tetanic stiumuation in the presence of omethoate and cyclohexanol. Voltage-clamp recordings of endplate currents (EPC) partially supported the EPP observations. Surprisingly, cyclohexanol-treated preparations showed no significant increase in EPC and MEPC decay time. However, there was some evidence of activity-dependent decline in MEPC amplitude in cyclohexanol while quantal content in these preparations showed evidence of an increase suggesting a homeostatic response in evoked transmitter release with cyclohexanol treatment. Analysis of presynaptic currents in cyclohexanol treated preparations also revealed preliminary evidence of sensitivity to cyclohexanol compared to control preparations. Finally, I tested the effects NMJ transmission of 24hr exposure to OP pesticide and its metabolites using a novel organ culture system, utilising a mouse mutant (WldS) with a slow nerve degeneration phenotype. After incubation of 24 hrs with MPS + pesticides and metabolites, these muscles showed significant reduction in function (response to nerve stimuli with EPP/action potential ± MEPPs) compared to control cultures. Together, the data indicate that failure of neuromuscular transmission by pesticide-plasma cannot be explained solely by dimethoate-mediated inhibition of acetylcholinesterase. Rather, a combination of metabolic breakdown products exerts potent, harmful presynaptic and postsynaptic effects. Either blocking the metabolic conversion of the constituents of OP pesticides, or transiently blocking their effects on receptors may therefore be an effective strategy for treatment of OP pesticide toxicity.

Neuropathische Schmerzen treten in der klinischen Praxis häufig auf und beeinträchtigen in hohem Maße die Lebensqualität der Patienten. Bedingt durch eine Schädigung somatosensorischer Nervenstrukturen im peripheren oder zentralen Nervensystem ist der Schmerz meist durch eine Allodynie, Hyperalgesie oder einschießende Schmerzen charakterisiert. Diese Symptome lassen sich durch gängige Schmerzmittel kaum lindern. In jüngerer Zeit rückte die Blut-Rückenmarkschranke immer mehr in den Fokus verschiedener Untersuchungen, da auch einige nicht-schmerzhafte Erkrankungen (z.B. Multiple Sklerose und Amyotrophe Lateralsklerose) zur Veränderung dieser Barriere mit folgender Permeabilitätserhöhung durch reduzierte Tight Junction-Protein-Expression führen. Die Blut- Rückenmarkschranke dichtet Gefäße des Rückenmarks ab und verhindert das Eindringen toxischer oder proanalgetischer Mediatoren in das zentrale Nervensystem. Dafür ist die Funktion der Tight Junction-Proteine zur Aufrechterhaltung dieser Barriere essentiell. Dennoch konnte die Rolle der Blut-Rückenmarkschranke bei Neuropathie noch nicht vollständig erörtert werden. Die Untersuchungen meiner Arbeitsgruppe konnten bereits zeigen, dass eine lockere Ligatur des N. ischiadicus bei Ratten (CCI; chronic constriction injury) zur Entwicklung einer thermischen und mechanischen Hyperalgesie sowie eingeschränkten motorischen Funktionen führt. Zudem konnte eine Störung der Blut-Rückenmarkschranke nach einer CCI nachgewiesen werden, da es Tracern unterschiedlicher molekularer Größe möglich war das Rückenmark zu penetrieren. Daher sollte im Rahmen dieser Dissertation untersucht werden, inwieweit es zu einer Veränderung unterschiedlicher Tight Junction-Proteine nach peripherer Nervenverletzung (CCI) kommt. Hierbei konnte gezeigt werden, dass eine CCI zu einer Herabregulation der mRNA-Expression von Claudin-1, Claudin-19, Tricellulin und Occludin im Rückenmark führt, wobei diese Veränderungen insbesondere 7 und 14 d nach der CCI auftraten. Die membranäre Expression dieser Proteine im Rückenmark blieb bis auf Occludin unverändert, das 7 d nach der CCI signifikant reduziert war. Am deutlichsten waren jedoch Claudin-5 und ZO-1 verändert. Folglich vermindert eine CCI signifikant die Claudin-5-mRNA sowie die Immunreaktivität in isolierten Kapillaren des Rückenmarks. Das Ankerprotein ZO-1 war sogar auf allen Ebenen, also in der Genals auch Proteinexpression, und darüber hinaus in den Rückenmarkskapillaren signifikant reduziert. Die Interpretation dieser Ergebnisse legt nahe, dass ZO-1, und zum Teil auch Claudin-5, für die gestörte Blut-Rückenmarkschranke verantwortlich sind und die anderen untersuchten Proteine vermutlich nur eine untergeordnete Rolle spielen. Die Bedeutung der Tight Junction-Proteine in der Blut-Rückenmarkschranke konnte somit weiter untermauert werden. In zukünftigen Untersuchungen wäre es wichtig den Signalweg, der zur Veränderung der Tight Junction-Proteine führt sowie die subzelluläre Lokalisation zu untersuchen, um Möglichkeiten zum Wiederverschluss der Barriere zu finden. Somit könnten Therapien zur Aufrechterhaltung der Blut- Rückenmarkhomöostase den neuropathischen Schmerz unter Umständen kausal, und nicht nur symptomatisch, behandelbar machen. / Chronic neuropathic pain is common in clinical practice and it greatly impairs the quality of life of patients. Due to a lesion or disease of the peripheral or central nervous system neuropathic pain is characterized by allodynia, hyperalgesia and spontaneous pain. The treatment of neuropathic pain remains a challenge since conventional pain treatment is not effective in alleviating most types of neuropathic pain. Lately a lot of research has focussed on the function of the blood-spinal cord barrier (BSCB) since some non-painful diseases (e.g. multiple sclerosis and amyotrophic lateral sclerosis) compromise the barrier and lead to reduced tight junction protein expression. The BSCB prevents leakage of molecules such as pronociceptive and toxic mediators into the spinal cord. Tight junction proteins are essential in maintaining this barrier by restricting the paracellular diffusion pathway. Nevertheless, the pathophysiology of neuropathic pain is not completely understood. Recently, this scientific work group observed that rats with a loose ligation of the sciatic nerve (CCI) developed thermal and mechanical hypersensitivity and reduced motor performance. The BSCB became permeable for small and large tracers indicating a breakdown of the barrier. This study was therefore designed to explore selected tight junction proteins after nerve injury using CCI. The mRNA expression of claudin-1, claudin-19, tricellulin and occludin was significantly reduced at 7 and 14 days after CCI. No alteration in protein expression was observed at any chosen time point except for occludin which was significantly decreased 7 days after CCI. In addition, CCI leads to a reduction of tight junction proteins most pronounced for claudin-5 an ZO-1. Claudin-5 mRNA expression and immunoreactivity in spinal cord capillaries is significantly reduced after CCI. The anchor protein ZO-1 is even downregulated at mRNA and protein levels in the whole lumbar spinal cord as well as in the spinal cord capillaries. In conclusion, this research shows that ZO-1 and in part claudin-5 are necessary for maintaining the BSCB while other tight junction proteins possibly play a minor role for the tightness of the barrier. These results further emphasize the importance of tight junction proteins in the BSCB. In future research the signaling pathway and regulation of these tight junction proteins should be explored as barrier sealing could be a possibility for pain relief in the future.

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We conducted 16 parallel in vitro selection experiments to isolate catalytic DNAs from a common DNA library for the cleavage of all 16 possible dinucleotide junctions of RNA incorporated into a common DNA/RNA chimeric substrate sequence. We discovered hundreds of sequence variations of the 8-17 deoxyribozyme - an RNA-cleaving catalytic DNA motif previously reported - from nearly all 16 final pools. Sequence analyses identified four absolutely conserved nucleotides in 8-17. Five representative 8-17 variants were tested for substrate cleavage in trans and together they were able to cleave 14 dinucleotide junctions. New 8-17 variants required Mn2+ to support their broad dinucleotide cleavage capabilities. We hypothesize that 8-17 has a tertiary structure composed of an enzymatic core executing catalysis and a structural facilitator providing structural fine-tuning when different dinucleotide junctions are given as cleavage sites. / Thesis / Master of Science (MSc)

Call number: LD2668 .T4 1963 J44 / Master of Science

ITC/USA 2007 Conference Proceedings / The Forty-Third Annual International Telemetering Conference and Technical Exhibition / October 22-25, 2007 / Riviera Hotel & Convention Center, Las Vegas, Nevada / Thermocouple measurements require “cold junction” compensation in order to obtain a correct reading. This compensation has traditionally been done with custom circuitry. In flight test applications where volume and power are at a premium (e.g. weapons flight test) it is desirable to have a more flexible solution that uses standard analog data acquisition channels already available as part of the encoder circuitry and performs compensation with remote software. This can be done via digital compensation, but certain measures must be taken to maintain accuracy and minimize noise. This paper describes some of these techniques and their performance tradeoffs.

Superconductor-Insulator-Superconductor (SIS) tunnel junction mixers are now commonly used in astronomical receivers at (sub)millimeter wavelengths because of their superb sensitivity, high dynamic range and stability of operation. Niobium SIS mixers operating at frequencies well below the super- conducting gap (∼680 GHz) have already achieved quantum limited sensitivity. Therefore to further enhance the receiver sensitivity, increasing the Intermediate Frequency (IF) bandwidth of SIS mixers has became crucial. This thesis focuses on the theoretical modeling, design and experimental verifi- cation of Nb SIS mixers operating around 230 GHz with a wide IF bandwidth of 1–15 GHz. These mixers were designed for a single baseline heterodyne interferometer (GUBBINS), which is being built to observe the Sunyaev-Zel’dovich effect in the Cosmic Microwave Background. The combination of wide IF bandwidth SIS mixers and complex analogue correlators will allow GUBBINS to feature high surface brightness sensitivity, that helps to distinguish the weak SZ effect from the background noise. The SIS mixer detector system was assembled inside the GUBBINS cryostat together with the IF electronics and RF/LO optical systems. Low noise temperatures of around 71 K were then measured in the GUBBINS system. The Nb SIS mixer we have developed uses a unilateral finline and fully integrated planar circuits deposited on a silicon substrate, to couple the electromagnetic radiation from the waveguide into the SIS junction. The finline mixer allows a broad-band RF coupling, an easy integration of the on-chip planar circuits and an easy-to-fabricate mixer block. To achieve a wide IF bandwidth, the output impedance of the SIS mixer was well matched to the input impedance of the amplifier by a multi-stage microstrip circuit. Additionally, the planar circuit of the SIS mixer was also designed to have a small lumped inductance and capacitance. The SIS mixer chip was extensively simulated by rigorous electromagnetic software (HFSS) and the S-parameter was exported to a quantum mixing package SuperMix to produce a full-wave model of the mixer. Experimental testing yielded a best noise temperature of 50 K with an average noise temperature of 75 K over an RF bandwidth of 160 GHz– 260 GHz. We have performed thorough experimental and computational investigation of the IF system in particular the constraints on the bandwidth caused by the lumped element capacitance of the mixer chip and the matching of the output impedance of the mixer to the IF amplifier. Our conclusion is that a bandwidth of 1–15 GHz could be achieved using our mixer design, subject to the performance of the amplifier. Finally, a variable temperature load system was successfully developed and tested inside the cryostat, to avoid the losses from the room-temperature optics. We have showed that the noise temperature of the SIS detector could be reduced by as much as 15 K by testing the mixer using a variable temperature load inside the cryostat.