Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-benzylated L-threo-β-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.

DNA probes for the detection of Fasciola hepatica in snails

Fasciola hepatica, also called the large liver fluke, is a trematode which can infect most mammals. Monitoring the infection rate of snails, which function as intermediate hosts and harbour larval stages of F. hepatica, is an important component of epidemiological studies on fascioliasis. For this purpose, DNA probes were generated which can be used for the detection of F. hepatica larvae in snails. Four highly repetitive DNA fragments were cloned in a plasmid vector and tested by Southern blot hybridization to the DNA of various trematodes for specificity and sensitivity. The probes Fhr-I, Fhr-II and Fhr-III hybridized only to F. hepatica DNA. Fhr-IV contained ribosomal RNA gene sequences and cross-hybridize with the DNA from various other trematode species. Squash blot analysis showed that the different probes were able to detect the parasite larvae in trematode-infected snails even as isolated single larvae. No signals were obtained in squash blots of uninfected snails. Probes Fhr-I, Fhr-II and Fhr-III are thus useful specific tools for studying the epidemiology of fascioliasis. The probe Fhr-IV, because of its broader spectrum, can be used to detect the larvae of a wide range of trematode species of waterbirds, which are the causative agents of swimmer's itch.

SYNTHETIC PROBES FOR STUDYING TUFTSIN-RECEPTOR INTERACTIONS ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES

Tuftsin is an immunopotentiating tetrapeptide of the sequence L-Thr-L-Lys-L-Pro-L-Arg with anti-microbial and anti-tumor enhancing capabilities. These enhancing functions are manifested through the host's granulocytes and monocytes. In delineating tuftsin's mechanism of action, both radiolabeled and fluorescent probes were synthesized. The radiolabeled probe of tuftsin, L-proly-3,4-('3)H(N) -tuftsin, was obtained through the synthesis and subsequent catalytic hydrogenation of L-3,4-dehydroprolyl ('3)-tuftsin using tritium gas. This procedure yielded a probe with a specific activity of 44.9 Ci/mmole. This radiolabeled probe of tuftsin was used in competitive inhibition studies with tuftsin, the tuftsin analogues Lys-Pro-Arg, Thr-Lys-Pro-Arg(NO(,2)) and (DELTA)('3)-pro('3) -tuftsin as well as with the chemotactic peptide f-Met-Leu-Phe. From the competitive binding curves, the K(,D) for tuftsin was estimated to be 80 nM, a value that approaches the concentration of tuftsin that evokes a half maximal biological response. The approximate Ki's for the tuftsin analogues (33 nM) approached that of tuftsin itself (40 nM). On the other hand, approximately a two log difference in the Ki was seen with the chemotactic tripeptide, indicating that tuftsin may indeed be acting through the chemotactic peptide receptor. This conclusion is further strengthened by studies using an N-terminal derivitized mono-fluoresceinated tuftsin probe and image intensification microscopy. These studies showed that like the chemotactic peptide, tuftsin initially binds to diffusely distributed receptors on the surface of human granulocytes. The tuftsin-receptor complexes then rapidly redistribute to form patches (5 min @ 37(DEGREES)C) which are then internalized. Whether redistribution and internalization of tuftsin-receptor complexes is crucial in effecting a biological response, or simply an intermediary point leading ultimately to degradation, is still not clear. This process, however, may provide the target cell with an early time point in modulating the biological effects of tuftsin through down-regulation of cell surface receptor sites. ^

(Table S2) Cell concentrations of individual and combined Alexandrium spp. and phylogenetic groups as obtained by qPCR assay and Utermöhl counts, as well as particulate PSP toxin concentrations from discrete water depths

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Interference of parallel cylindrical Langmuir probes

Current to a cylindrical probe of arbitrary cross section is discussed. Previous results for circular cylinders at the high bias and moderate radius R of interest for electrodynamic bare tethers, for which space charge may be ignored over a large neighborhood of the probe, depend in separate ways on both R and perimeter p. These results are extended to a general convex cross section by introducing certain equivalent radius Req. For any concave cross section, results use a proper equivalent perimeter peq , in addition to Req. Finally, for the joint cross section of separate parallel probes, certain effective perimeter peff replaces peq. Rules to determine Req. peq. and peff are used to discuss collection interference among two or more parallel cylinders when brought from far away to contact

The orbital-motion-limited regime of cylindrical Langmuir probes

An asymptotic analysis of electron collection at high bias Fp serves to determine the domain of validity of the orbital-motion-limited regime of cylindrical Langmuir probes, which is basic for the workings of conductive bare tethers. The radius of a wire collecting OML current in an unmagnetized plasma at rest cannot exceed a value, Rmax , which is found to exhibit a minimum as a function of Fp ; atFp values of interest, Rmax is already increasing and is larger than the electron Debye length lDe . The breakdown of the regime relates to conditions far fromthe probe, at electron energies comparable to the ion thermal energy, kTi ; Rmax is found to increase with Ti . It is also found that ~1! the maximumwidth of a thin tape, if used instead of a wire, is 4Rmax ; ~2! the electron thermal gyroradius must be larger than both R and lDe for magnetic effects to be negligible; and ~3! conditions applying to the tether case are such that trapped-orbit effects are negligible.

Cylindrical Langmuir probes beyond the orbital-motion-limited regime

The current I to a cylindrical probe at rest in an unmagnetized plasma, with probe bias highly positive, is determined. The way I lags behind the orbital-motion-limited OMLcurrent, 1 OML R, as the radius R exceeds the maximum radius for the OML regime to hold, is of interest for space-tether applications. The ratio I/I OML is roughly a decreasing function of R/lD R max /lDe , which is independent of bias, with lDe the electron Debye length and Rmax /l De roughly an increasing function of the temperature ratio, Ti /Te. The dependence of current on ion energy is used to discuss the effect of probe motion through the plasma, a case applying to tethers in low orbit.

The theory of electrostatic probes in strong magnetic fields : Thesis submitted to the Faculty of The Graduate School of the University of Colorado for the Degree Doctor of Philosophy Department of Aerospace Engineering Science

A theory is developed of an electrostatic probe in a fully-ionized plasma in the presence of a strong magnetic field. The ratio of electron Larmor radius to probe transverse dimension is assumed to be small. Poisson's equation, together with kinetic equations for ions and electrons are considered. An asymptotic perturbation method of multiple scales is used by considering the characteristic lengths appearing in the problem. The leading behavior of the solution is found. The results obtained appear to apply to weaker fields also, agreeing with the solutions known in the limit of no magnetic field. The range of potentials for wich results are presented is limited. The basic effects produced by the field are a depletion of the plasma near the probe and a non-monotonic potential surrounding the probe. The ion saturation current is not changed but changes appear in both the floating potential Vf and the slope of the current-voltage diagram at Vf. The transition region extends beyond the space potential Vs,at wich point the current is largely reduced. The diagram does not have an exponential form in this region as commonly assumed. There exists saturation in electron collection. The extent to which the plasma is disturbed is determined. A cylindrical probe has no solution because of a logarithmic singularity at infinity. Extensions of the theory are considered.

Emissive Langmuir Probes in the Strong Emission Regime for the Determination of the Plasma Properties

The determination of the plasma potential Vpl of unmagnetized plasmas by using the floating potential of emissive Langmuir probes operated in the strong emission regime is investigated. The experiments evidence that, for most cases, the electron thermionic emission is orders of magnitude larger than the plasma thermal electron current. The temperature-dependent floating potentials of negatively biased Vpmenor queVpl emissive probes are in agreement with the predictions of a simple phenomenological model that considers, in addition to the plasma electrons, an ad-ditional electron group that contributes to the probe current. The latter would be constituted by a fraction of the repelled electron thermionic current, which might return back to the probe with a different energy spectrum. Its origin would be a plasma potential well formed in the plasma sheath around the probe, acting as a virtual cathode or by collisions and electron thermalization pro-cesses. These results suggest that, for probe bias voltages close to the plasma potential Vp?Vpl, two electron populations coexist, i.e., the electrons from the plasma with temperatureTeand a large group of returned thermionic electrons. These results question the theoretical possibility of measuring the electron temperature by using emissive probes biased to potentials Vp about lower equal than ?Vpl.

Diagnostics of classical and dusty plasmas w ith collecting and emissive langmuir probes = Diagnosis de plasmas clásicos y granulares mediante sondas colectoras y emisoras de Langmuir

Las sondas eléctricas se emplean habitualmente en la diagnosis de plasmas. La presente tesis aborda la operación de las sondas colectoras y emisoras de Langmuir en plasmas fríos de baja densidad. El estudio se ha centrado en la determinación del potencial de plasma, Vsp, mediante el potencial flotante de una sonda emisora. Esta técnica consiste en la medida del potencial de la sonda correspondiente a la condición de corriente neta igual a cero, el cual se denomina potencial flotante, VF. Este potencial se desplaza hacia el potencial del plasma según aumenta la emisión termoiónica de la sonda, hasta que se satura cerca de Vsp. Los experimentos llevados a cabo en la pluma de plasma de un motor iónico y en un plasma de descarga de glow muestran que la corriente de electrones termoiónicos es mayor que la corriente de electrones recogidos para una sonda polarizada por debajo del potencial del plasma, resultado inconsistente con la teoría tradicionalmente aceptada. Para investigar estos resultados se ha introducido el parámetro R, definido como el cociente entre la corriente de electrones emitidos y recogidos por la sonda. Este parámetro, que está relacionado con la diferencia de potencial VF - Vsp, también es útil para la descripción de los modos de operación de la sonda emisora (débil, fuerte y más allá del fuerte). Los resultados experimentales evidencian que, al contrario de lo que indica la teoría, R es mayor que la unidad. Esta discrepancia se puede solucionar introduciendo una población efectiva de electrones. Con dicha población, el nuevo modelo para la corriente total de la sonda reproduce los datos experimentales. El origen de este grupo electrónico es todavía una cuestión abierta, pero podría estar originada por una nueva estructura de potencial cerca de la sonda cuando ésta trabaja en el régimen de emisión fuerte. Para explicar dicha estructura de potencial, se propone un modelo unidimensional compuesto por un mínimo de potencial cerca de la superficie de la sonda. El análisis numérico indica que este pozo de potencial aparece para muy altas temperaturas de la sonda, reduciendo la cantidad de electrones emitidos que alcanzan el plasma y evitando así cualquier posible perturbación de éste. Los aspectos experimentales involucrados en el método del potencial flotante también se han estudiado, incluyendo cuestiones como las diferentes técnicas de obtención del VF, el cociente señal-ruido, el acoplamiento de la señal de los equipos utilizados para la obtención de las curvas I-V o la evidencia experimental de los diferentes modos de operación de la sonda. Estas evidencias empíricas se encuentran en todos los aspectos de operación de la sonda: la recolección de electrones, el potencial flotante, la precisión en las curvas I-V y la emisión electrónica. Ésta última también se estudia en la tesis, debido a que un fenómeno de super emisión tiene lugar en el régimen de emisión fuerte. En este modo de operación, las medidas experimentales indican que las corrientes termoiónicas de electrones son mayores que aquéllas predichas por la ecuación de Richardson-Dushman clásica. Por último, la diagnosis de plasmas usando sondas eléctrica bajo presencia de granos de polvo (plasmas granulares) en plasmas fríos de baja densidad también se ha estudiado, mediante la aplicación numérica de la técnica del potencial flotante de la sonda emisora en un plasma no convencional. Los resultados apuntan a que el potencial flotante de una sonda emisora se vería afectado por altas densidades de polvo o grandes partículas. ABSTRACT Electric probes are widely employed for plasma diagnostics. This dissertation concerns the operation of collecting and emissive Langmuir probes in low density cold plasmas. The study is focused on the determination of the plasma potential, Vsp, by means of the floating potential of emissive probes. This technique consists of the measurement of the probe potential, corresponding to the zero net probe current, which is the so-called floating potential, VF . This potential displaces towards the plasma potential as the thermionic electron emission increases, until it saturates near Vsp. Experiments carried out in the plasma plume of an ion thruster and in a glow discharge plasma show the thermionic electron current of the emissive Langmuir probe is higher than the collected electron current, for a probe with a bias potential below Vsp, which is inconsistent with the traditional accepted theory. To investigate these results, a parameter R is introduced as the ratio between the emitted and the collected electron current. This parameter, which is related to the difference VF - Vsp, is also useful for the description of the operation modes of the emissive Langmuir probe (weak, strong and beyond strong). The experimental results give an inconsistency of R > 1, which is solved by a modification of the theory for emissive probes, with the introduction of an effective electron population. With this new electron group, the new model for the total probe current agrees with the experimental data. The origin of this electron group remains an open question, but it might be originated by a new potential structure near the emissive probe when it operates in the strong emission regime. A simple one-dimension model composed by a minimum of potential near the probe surface is discussed for strongly emitting emissive probes. The results indicate that this complex potential structure appears for very high probe temperatures and the potential well might reduce the emitted electrons population reaching the plasma bulk. The experimental issues involved in the floating potential method are also studied, as the different obtaining techniques of VF, the signal-to-noise ratio, the signal coupling of the I-V curve measurement system or the experimental evidence of the probe operation modes. These empirical proofs concern all the probe operation aspects: the electron collection, the floating potential, the I-V curve accuracy as well as the electron emission. This last issue is also investigated in this dissertation, because a super emission takes place in the strong emission regime. In this operation mode, the experimental results indicate that the thermionic electron currents might be higher than those predicted by the classical Richardson-Dushman equation. Finally, plasma diagnosis using electric probes in the presence of dust grains (dusty plasmas) in low density cold plasmas is also addressed. The application of the floating potential technique of the emissive probe in a non-conventional complex plasma is numerically investigated, whose results point out the floating potential of the emissive probe might be shifted for high dust density or large dust particles.

Direct Vlasov simulations of electron-attracting cylindrical Langmuir probes in flowing plasmas

Current collection by positively polarized cylindrical Langmuir probes immersed in flowing plasmas is analyzed using a non-stationary direct Vlasov-Poisson code. A detailed description of plasma density spatial structure as a function of the probe-to-plasma relative velocity U is presented. Within the considered parametric domain, the well-known electron density maximum close to the probe is weakly affected by U. However, in the probe wake side, the electron density minimum becomes deeper as U increases and a rarified plasma region appears. Sheath radius is larger at the wake than at the front side. Electron and ion distribution functions show specific features that are the signature of probe motion. In particular, the ion distribution function at the probe front side exhibits a filament with positive radial velocity. It corresponds to a population of rammed ions that were reflected by the electric field close to the positively biased probe. Numerical simulations reveal that two populations of trapped electrons exist: one orbiting around the probe and the other with trajectories confined at the probe front side. The latter helps to neutralize the reflected ions, thus explaining a paradox in past probe theory.