Electrostatic sampling of trace DNA from clothing.

During acts of physical aggression, offenders frequently come into contact with clothes of the victim, thereby leaving traces of DNA-bearing biological material on the garments. Since tape-lifting and swabbing, the currently established methods for non-destructive trace DNA sampling from clothing, both have their shortcomings in collection efficiency and handling, we thought about a new collection method for these challenging samples. Testing two readily available electrostatic devices for their potential to sample biological material from garments made of different fabrics, we found one of them, the electrostatic dust print lifter (DPL), to perform comparable to well-established sampling with wet cotton swabs. In simulated aggression scenarios, we had the same success rate for the establishment of single aggressor profiles, suitable for database submission, with both the DPL and wet swabbing. However, we lost a substantial amount of information with electrostatic sampling, since almost no mixed aggressor-victim profiles suitable for database entry could be established, compared to conventional swabbing. This study serves as a proof of principle for electrostatic DNA sampling from items of clothing. The technique still requires optimization before it might be used in real casework. But we are confident that in the future it could be an efficient and convenient contribution to the toolbox of forensic practitioners.

Prevention of vascular dysfunction and arterial hypertension in mice generated by assisted reproductive technologies by addition of melatonin to culture media.

Assisted reproductive technologies (ART) induce vascular dysfunction in humans and mice. In mice, ART-induced vascular dysfunction is related to epigenetic alteration of the endothelial nitric oxide synthase (eNOS) gene, resulting in decreased vascular eNOS expression and nitrite/nitrate synthesis. Melatonin is involved in epigenetic regulation, and its administration to sterile women improves the success rate of ART. We hypothesized that addition of melatonin to culture media may prevent ART-induced epigenetic and cardiovascular alterations in mice. We, therefore, assessed mesenteric-artery responses to acetylcholine and arterial blood pressure, together with DNA methylation of the eNOS gene promoter in vascular tissue and nitric oxide plasma concentration in 12-wk-old ART mice generated with and without addition of melatonin to culture media and in control mice. As expected, acetylcholine-induced mesenteric-artery dilation was impaired (P = 0.008 vs. control) and mean arterial blood pressure increased (109.5 ± 3.8 vs. 104.0 ± 4.7 mmHg, P = 0.002, ART vs. control) in ART compared with control mice. These alterations were associated with altered DNA methylation of the eNOS gene promoter (P < 0.001 vs. control) and decreased plasma nitric oxide concentration (10.1 ± 11.1 vs. 29.5 ± 8.0 μM) (P < 0.001 ART vs. control). Addition of melatonin (10(-6) M) to culture media prevented eNOS dysmethylation (P = 0.005, vs. ART + vehicle), normalized nitric oxide plasma concentration (23.1 ± 14.6 μM, P = 0.002 vs. ART + vehicle) and mesentery-artery responsiveness to acetylcholine (P < 0.008 vs. ART + vehicle), and prevented arterial hypertension (104.6 ± 3.4 mmHg, P < 0.003 vs. ART + vehicle). These findings provide proof of principle that modification of culture media prevents ART-induced vascular dysfunction. We speculate that this approach will also allow preventing ART-induced premature atherosclerosis in humans.

Verification of intensity modulated stereotactic radiotherapy using Monte Carlo calculations and EPID dosimetry

The purpose of this work was to develop a comprehensive IMSRT QA procedure that examined, using EPID dosimetry and Monte Carlo (MC) calculations, each step in the treatment planning and delivery process. These steps included verification of the field shaping, treatment planning system (RTPS) dose calculations, and patient dose delivery. Verification of each step in the treatment process is assumed to result in correct dose delivery to the patient. ^ The accelerator MC model was verified against commissioning data for field sizes from 0.8 × 0.8 cm 2 to 10 × 10 cm 2. Depth doses were within 2% local percent difference (LPD) in low gradient regions and 1 mm distance to agreement (DTA) in high gradient regions. Lateral profiles were within 2% LPD in low gradient regions and 1 mm DTA in high gradient regions. Calculated output factors were within 1% of measurement for field sizes ≥1 × 1 cm2. ^ The measured and calculated pretreatment EPID dose patterns were compared using criteria of 5% LPD, 1 mm DTA, or 2% of central axis pixel value with ≥95% of compared points required to pass for successful verification. Pretreatment field verification resulted in 97% percent of the points passing. ^ The RTPS and Monte Carlo phantom dose calculations were compared using 5% LPD, 2 mm DTA, or 2% of the maximum dose with ≥95% of compared points required passing for successful verification. RTPS calculation verification resulted in 97% percent of the points passing. ^ The measured and calculated EPID exit dose patterns were compared using criteria of 5% LPD, 1 mm DTA, or 2% of central axis pixel value with ≥95% of compared points required to pass for successful verification. Exit dose verification resulted in 97% percent of the points passing. ^ Each of the processes above verified an individual step in the treatment planning and delivery process. The combination of these verification steps ensures accurate treatment delivery to the patient. This work shows that Monte Carlo calculations and EPID dosimetry can be used to quantitatively verify IMSRT treatments resulting in improved patient care and, potentially, improved clinical outcome. ^

The survival role and the potential therapeutic targeting of MET in multiple myeloma

Multiple myeloma (MM) is a debilitating and incurable B-cell malignancy. Previous studies have documented that the hepatocyte growth factor (HGF) plays a role in the pathobiology of MM. The receptor tyrosine kinase MET induced signaling initiates when its ligand HGF binds to the MET receptor. However, the direct importance of MET in MM has not been elucidated. The present work used three different but complementary approaches to reduce MET protein levels or its activity to demonstrate the importance of MET in MM. ^ In the first approach, MET transcript and protein levels were reduced by directly targeting the cellular MET transcripts using shRNA retroviral infection techniques. This direct reduction of MET mRNA leads to a reduction of MET protein levels, which caused an inhibition of growth and induction of cell death. ^ In the second approach, a global transcription inhibitor flavopiridol was used as a potential pharmacological tool to reduce MET levels. MET has a short half-life of 30 min for mRNA and 4 hours for protein; therefore using a RNA pol II inhibitor such as flavopiridol would be a viable option to reduce MET levels. When using flavopiridol in MM cell lines, there was a reduction of MET transcript and protein levels, which was associated with the induction of cell death. ^ Finally in the last strategy, MET kinase activity was suppressed by MP470, a small molecule inhibitor that binds to the ATP binding pocket in the kinase domain. At concentrations where phosphorylation of MET was inhibited there was induction of cell death in MM cell lines and primary cells from patients. In addition, in MM cell lines there was a decrease in phosphorylation of AKT (ser473) and caspase-9 (ser196); downstream of MET, suggesting that the mechanism of action for survival may be through these cascade of events. ^ Overall, this study provides a proof-of-principle that MET is important for the survival of MM cell lines as well as primary plasma cells obtained from patients. Therefore, targeting MET therapeutically may be a possible strategy to treat patients with this debilitating disease of MM. ^

ECONOMIC ANALYSIS OF THE DEMAND FOR PRESCRIPTION MEDICINE IN THE UNITED STATES

The purpose of this study was to understand the role of principle economic, sociodemographic and health status factors in determining the likelihood and volume of prescription drug use. Econometric demand regression models were developed for this purpose. Ten explanatory variables were examined: family income, coinsurance rate, age, sex, race, household head education level, size of family, health status, number of medical visits, and type of provider seen during medical visits. The economic factors (family income and coinsurance) were given special emphasis in this study.^ The National Medical Care Utilization and Expenditure Survey (NMCUES) was the data source. The sample represented the civilian, noninstitutionalized residents of the United States in 1980. The sample method used in the survey was a stratified four-stage, area probability design. The sample was comprised of 6,600 households (17,123 individuals). The weighted sample provided the population estimates used in the analysis. Five repeated interviews were conducted with each household. The household survey provided detailed information on the United States health status, pattern of health care utilization, charges for services received, and methods of payments for 1980.^ The study provided evidence that economic factors influenced the use of prescription drugs, but the use was not highly responsive to family income and coinsurance for the levels examined. The elasticities for family income ranged from -.0002 to -.013 and coinsurance ranged from -.174 to -.108. Income has a greater influence on the likelihood of prescription drug use, and coinsurance rates had an impact on the amount spent on prescription drugs. The coinsurance effect was not examined for the likelihood of drug use due to limitations in the measurement of coinsurance. Health status appeared to overwhelm any effects which may be attributed to family income or coinsurance. The likelihood of prescription drug use was highly dependent on visits to medical providers. The volume of prescription drug use was highly dependent on the health status, age, and whether or not the individual saw a general practitioner. ^

Posttraumatic stress disorder among veterans of the Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF) wars: Psychological comorbidities, postdeployment social support and unit support

Much attention has been given to treating Operation Iraqi Freedom/Operation Enduring (OIF/OEF) Veterans with posttraumatic stress disorder (PTSD). However, little attention is given to those Veterans who do not meet diagnostic criteria for PTSD but who may still benefit from intervention. Research is needed to investigate the impact of how different racial/ethnic backgrounds, different levels of social support and comorbid mental health disorders impact OIF/OEF Veterans with varying levels of PTSD. The purpose of this dissertation is to examine the association of comorbid Axis I disorders, race/ethnicity, different levels of postdeployment social support and unit support on OIF/OEF Veterans with varying levels of PTSD. Data for this dissertation were from postdeployment screenings of OIF/OEF Veterans from a large Veterans Affairs hospital in southeast Texas. To examine the study hypotheses, we conducted multinomial logistic regressions of the clinician reported data. ^ The first article examined the prevalence of subthreshold and full levels of PTSD and compared Axis I and alcohol use comorbidity rates among 1,362 OIF/OEF Veterans with varying levels of PTSD. Results suggest that OIF/OEF Veterans with subthreshold PTSD experience similar levels of psychological distress as those with full PTSD and highlight the need to provide timely and appropriate mental health services to individuals who may not meet the diagnostic criteria for full PTSD. ^ These results suggest that OIF/OEF Veterans of all race/ethnicities can benefit from strong social support systems. Postdeployment social support was found to be a protective factor against the development of PTSD among White, Black and Hispanic veterans while deployment unit support was a protective factor only among Black Veterans. The second article investigated the association between postdeployment social support and unit support with varying levels of PTSD by race/ethnicity among 1,115 OIF/OEF Veterans. ^ The results of this study can help to formulate treatment and interventions for OIF/OEF Veterans with varying levels of PTSD and social support systems.^

Predictors of benzodiazepine self -administration behavior in anxious patients

The purpose of this study was to examine factors that may be associated with benzodiazepine (BZ) self-administration and risks of dependence in anxious patients. Preliminary work included examination of psychosocial characteristics and subjective drug response as potential predictors of medication use. Fifty-five M, F patients with generalized anxiety or panic disorder participated in a 3-week outpatient Choice Procedure in which they self-medicated “as needed” with alprazolam (Alz) and placebo. Findings showed that a large amount of variance in alprazolam preference, frequency, and quantity of use could be predicted by measures of anxiety, drug liking, and certain personality characteristics. The primary study extended this work by examining whether individual differences in Alz sensitivity also predict patterns of use. Twenty anxious patients participated in the study, which required 11 weekly clinic visits. Ten of these also participated in a baseline assessment of HPA-axis function that involved 24-hour monitoring of cortisol and ACTH levels and a CRH Stimulation Test. This assessment was conducted on the basis of prior evidence that steroid metabolites exert neuromodulatory effects on the GABA A receptor and that HPA-axis function may be related to BZ sensitivity and long-term disability in anxious patients. Patients were classified as either HIGH or LOW users based on their p.r.n. patterns of Alz use during the first 3 weeks of the study. They then participated in a 4-week dose response trial in which they received prescribed doses of medication (placebo, 0.25, 0.5, and 1.0mg Alz), each taken TID for 1 week. The dose response trial was followed by a second 3-week Choice Procedure. Findings were not indicative of biological differences in Alz sensitivity between the HIGH and LOW users. However, the HIGH users had higher baseline anxiety and greater anxiolytic response to Alz than the LOW users. Anxiolytic benefits of p.r.n. and prescribed dosing were shown to be comparable, and patients' conservative patterns of p.r.n. medication use were not affected by the period of prescribed dosing. Although there was not strong evidence to suggest relationships between HPA-axis function and Alz use or sensitivity, interesting findings emerged about the relationship between HPA-axis function and anxiety. ^

Stable oxygen isotope record of epibenthic foraminifera and water samples of the Arctic Ocean

We determined d18OCib values of live (Rose Bengal stained) and dead epibenthic foraminifera Cibicidoides wuellerstorfi, Cibicides lobatulus, and Cibicides refulgens in surface sediment samples from the Arctic Ocean and the Greenland, Iceland, and Norwegian seas (Nordic Sea). This is the first time that a comprehensive d18OCib data set is generated and compiled from the Arctic Ocean. For comparison, we defined Atlantic Water (AW), upper Arctic Bottom Water (uABW), and Arctic Bottom Water (ABW) by their temperature/salinity characteristics and calculated mean equilibrium calcite d18Oequ from summer sea-water d18Ow and in situ temperatures. As a result, in the Arctic environment we compensate for Cibicidoides- and Cibicides-specific offsets from equilibrium calcite of -0.35 and -0.55 per mil, respectively. After this taxon-specific adjustment, mean d18OCib values plausibly reflect the density stratification of principle water masses in the Nordic Sea and Arctic Ocean. In addition, mean d18OCib from AW not only significantly differs from mean d18OCib from ABW, but also d18OCib from within AW differentiates in function of provenience and water mass age. Furthermore, in shallow waters brine-derived low d18Ow can significantly lower the d18OCib of Cibicides spp. and thus d18OCib may serve as a paleobrine indicator. There is no statistically significant difference, however, between deeper water masses mean d18OCib of the Nordic Sea, and of the Eurasian and Amerasian basins, and no influence of low-d18Ow brines is recorded in Recent uABW and ABW d18OCib of C. wuellerstorfi. This may be due to dilution of a low-d18Ow brine signal in the deep sea, and/or to preferential incorporation of relatively high-d18Ow brines from high-salinity shelves. Although our data encompass environments with seasonal sea-ice and brine formation supposed to ultimately ventilate the deep Arctic Ocean, d18OCib from uABW and ABW do not indicate negative excursions. This may challenge hypotheses that call for enhanced Arctic brine release to explain negative benthic d18O spikes in deep-sea sediments from the late Pleistocene North Atlantic Ocean.

Paleomagnetic of lower Cretaceous sediments from the Argo and Gascoyne Abyssal Plain

Lower Cretaceous sediments were sampled for magnetostratigraphy at three sites. ODP Site 765 and DSDP Site 261, in the Argo Abyssal Plain, consist primarily of brownish-red to gray claystone having hematite and magnetite carriers of characteristic magnetization. ODP Site 766, in the Gascoyne Abyssal Plain, consists mainly of dark greenish-gray volcaniclastic turbidites with magnetite as the carrier of characteristic magnetization. Progressive thermal demagnetization (Sites 765 and 261) or alternating field demagnetization (Site 766) yielded well-defined polarity zones and a set of reliable paleolatitudes. Magnetic polarity chrons were assigned to polarity zones using biostratigraphic correlations. Late Aptian chron M"-1"r, a brief reversed-polarity chron younger than MOr, is a narrow, 40-cm feature delimited in Hole 765C. Early Aptian reversed-polarity chron MOr is also present in Hole 765C. Polarity chrons Mir through M3r were observed in the Barremian of all three sites. Valanginian and Hauterivian polarity chrons can be tentatively assigned to polarity zones only in Hole 766A. The paleolatitude of this region remained at 35° to 37°S from the Berriasian through the Aptian. During this interval, there was approximately 16° of clockwise rotation, with the oriented sample suites of Site 765 displaying a Berriasian declination of 307° to an Aptian declination of 323°. These results are consistent with the interpolated Early Cretaceous apparent polar wander for Australia, but indicate that this region was approximately 5? farther north than predicted.

137Cs and 90Sr depositions, and 137Cs/90Sr ratios of samples from the Kraton-3 underground nuclear explosion site, Russia

Strontium-90 activity concentrations in surface soils and areal deposition densities have been studied at a site contaminated by an accidental release to atmosphere from the underground nuclear explosion 'Kraton-3' conducted near the Polar Circle (65.9°N, 112.3°E) within the territory of the former USSR in 1978. In 2001-2002, the ground surface contamination at 14 plots studied ranged from 20 to 15000 kBq/m**2, which significantly exceeds the value of 0.44 kBq/m**2 deduced for three background plots. The zone with substantial radiostrontium contamination extends, at least, 2.5 km in a north-easterly direction from the borehole. The average 137Cs/90Sr ratio in the ground contamination originated from the 'Kraton-3' fallout was estimated to be 0.55, which is significantly different from the ratio of 2.05 evaluated for background plots contaminated mostly from global fallout. Although vertical migration of 90Sr in all undisturbed soil profiles studied is more rapid than that for 137Cs, the depth of percolation of both radionuclides into the ground is mostly limited to the top 10-20 cm, which may be explained, primarily, by permafrost conditions. The horizontal migration rate of radiostrontium in the aqueous phase exceeds the radiocaesium migration rate by many times. This phenomenon seems to be a reason for the significant enrichment of the soil surface layers by radiostrontium at some sites, with variations occurring in accordance with small-scale irregularities of landscape.

Effect of substrate-target distance and sputtering pressure in the synthesis of AlN thin films

In this work, we analyze the influence of the processing pressure and the substrate–target distance on the synthesis by reactive sputtering of c-axis oriented polycrystalline aluminum nitride thin films deposited on Si(100) wafers. The crystalline quality of AlN has been characterized by high-resolution X-ray diffraction (HR-XRD). The films exhibited a very high degree of c-axis orientation especially when a low process pressure was used. After growth, residual stress measurements obtained indirectly from radius of curvature measurements of the wafer prior and after deposition are also provided. Two different techniques are used to determine the curvature—an optically levered laser beam and a method based on X-ray diffraction. There is a transition from compressive to tensile stress at a processing pressure around 2 mTorr. The transition occurs at different pressures for thin films of different thickness. The degree of c-axis orientation was not affected by the target–substrate distance as it was varied in between 30 and 70 mm.

Analytical models for the power subsystem and the attitude control subsystem of a microsatellite

El trabajo realizado en la presente tesis doctoral se debe considerar parte del proyecto UPMSat-2, que se enmarca dentro del ámbito de la tecnología aeroespacial. El UPMSat-2 es un microsatélite (de bajo coste y pequeño tamaño) diseñado, construido, probado e integrado por la Universidad Politécnica de Madrid (España), para fines de demostración tecnológica y educación. El objetivo de la presente tesis doctoral es presentar nuevos modelos analíticos para estudiar la interdependencia energética entre los subsistemas de potencia y de control de actitud de un satélite. En primer lugar, se estudia la simulación del subsistema de potencia de un microsatélite, prestando especial atención a la simulación de la fuente de potencia, esto es, los paneles solares. En la tesis se presentan métodos sencillos pero precisos para simular la producción de energía de los paneles en condiciones ambientales variables a través de su circuito equivalente. Los métodos propuestos para el cálculo de los parámetros del circuito equivalente son explícitos (o al menos, con las variables desacopladas), no iterativos y directos; no se necesitan iteraciones o valores iniciales para calcular los parámetros. La precisión de este método se prueba y se compara con métodos similares de la literatura disponible, demostrando una precisión similar para mayor simplicidad. En segundo lugar, se presenta la simulación del subsistema de control de actitud de un microsatélite, prestando especial atención a la nueva ley de control propuesta. La tesis presenta un nuevo tipo de control magnético es aplicable a la órbita baja terrestre (LEO). La ley de control propuesta es capaz de ajustar la velocidad de rotación del satélite alrededor de su eje principal de inercia máximo o mínimo. Además, en el caso de órbitas de alta inclinación, la ley de control favorece la alineación del eje de rotación con la dirección normal al plano orbital. El algoritmo de control propuesto es simple, sólo se requieren magnetopares como actuadores; sólo se requieren magnetómetros como sensores; no hace falta estimar la velocidad angular; no incluye un modelo de campo magnético de la Tierra; no tiene por qué ser externamente activado con información sobre las características orbitales y permite el rearme automático después de un apagado total del subsistema de control de actitud. La viabilidad teórica de la citada ley de control se demuestra a través de análisis de Monte Carlo. Por último, en términos de producción de energía, se demuestra que la actitud propuesto (en eje principal perpendicular al plano de la órbita, y el satélite que gira alrededor de ella con una velocidad controlada) es muy adecuado para la misión UPMSat-2, ya que permite una área superior de los paneles apuntando hacia el sol cuando se compara con otras actitudes estudiadas. En comparación con el control de actitud anterior propuesto para el UPMSat-2 resulta en un incremento de 25% en la potencia disponible. Además, la actitud propuesto mostró mejoras significativas, en comparación con otros, en términos de control térmico, como la tasa de rotación angular por satélite puede seleccionarse para conseguir una homogeneización de la temperatura más alta que apunta satélite y la antena. ABSTRACT The work carried out in the present doctoral dissertation should be considered part of the UPMSat-2 project, falling within the scope of the aerospace technology. The UPMSat-2 is a microsatellite (low cost and small size) designed, constructed integrated and tested for educational and technology demonstration purposes at the Universidad Politécnica de Madrid (Spain). The aim of the present doctoral dissertation is to present new analytical models to study the energy interdependence between the power and the attitude control subsystems of a satellite. First, the simulation of the power subsystem of a microsatellite is studied, paying particular attention to the simulation of the power supply, i.e. the solar panels. Simple but accurate methods for simulate the power production under variable ambient conditions using its equivalent circuit are presented. The proposed methods for calculate the equivalent circuit parameters are explicit (or at least, with decoupled variables), non-iterative and straight forward; no iterations or initial values for the parameters are needed. The accuracy of this method is tested and compared with similar methods from the available literature demonstrating similar precision but higher simplicity. Second, the simulation of the control subsystem of a microsatellite is presented, paying particular attention to the new control law proposed. A new type of magnetic control applied to Low Earth Orbit (LEO) satellites has been presented. The proposed control law is able to set the satellite rotation speed around its maximum or minimum inertia principal axis. Besides, the proposed control law favors the alignment of this axis with the normal direction to the orbital plane for high inclination orbits. The proposed control algorithm is simples, only magnetorquers are required as actuators; only magnetometers are required as sensors; no estimation of the angular velocity is needed; it does not include an in-orbit Earth magnetic field model; it does not need to be externally activated with information about the orbital characteristics and it allows automatic reset after a total shutdown of attitude control subsystem. The theoretical viability of the control law is demonstrated through Monte Carlo analysis. Finally, in terms of power production, it is demonstrated that the proposed attitude (on principal axis perpendicular to the orbit plane, and the satellite rotating around it with a controlled rate) is quite suitable for the UPMSat-2 mission, as it allows a higher area of the panels pointing towards the sun when compared to other studied attitudes. Compared with the previous attitude control proposed for the UPMSat-2 it results in a 25% increment in available power. Besides, the proposed attitude showed significant improvements, when compared to others, in terms of thermal control, as the satellite angular rotation rate can be selected to achieve a higher temperature homogenization of the satellite and antenna pointing.

Design, Technology and Characterization of Micromechanised Sensors and Actuators for Harsh Environments

Los sistemas micro electro mecánicos (MEMS) han demostrado ser una exitosa familia de dispositivos que pueden usarse como plataforma para el desarrollo de dispositivos con aplicaciones en óptica, comunicaciones, procesado de señal y sensorización. Los dispositivos MEMS estándar suelen estar fabricados usando tecnología de silicio. Sin embargo, el rendimiento de estos MEMS se puede mejorar si se usan otros materiales. Por ejemplo, el diamante nanocristalino (NCD) ofrece unas excelentes propiedades mecánicas, transparencia y una superficie fácil de funcionalizar. Por otro lado, el sistema de materiales (In; Ga; Al)N, los materiales IIIN, se pueden usar para producir estructuras monocristalinas con alta sensibilidad mecánica y química. Además, el AlN se puede depositar por pulverización catódica reactiva sobre varios substratos, incluyendo NCD, para formar capas policristalinas orientadas con alta respuesta piezoeléctrica. Adicionalmente, tanto el NCD como los materiales III-N muestran una gran estabilidad térmica y química, lo que los hace una elección idónea para desarrollar dispositivos para aplicaciones para alta temperatura, ambientes agresivos e incluso para aplicaciones biocompatibles. En esta tesis se han usado estos materiales para el diseño y medición de demostradores tecnológicos. Se han perseguido tres objetivos principales: _ Desarrollo de unos procesos de fabricación apropiados. _ Medición de las propiedades mecánicas de los materiales y de los factores que limitan el rendimiento de los dispositivos. _ Usar los datos medidos para desarrollar dispositivos demostradores complejos. En la primera parte de esta tesis se han estudiado varias técnicas de fabricación. La estabilidad de estos materiales impide el ataque y dificulta la producción de estructuras suspendidas. Los primeros capítulos de esta disertación se dedican al desarrollo de unos procesos de transferencia de patrones por ataque seco y a la optimización del ataque húmedo sacrificial de varios substratos propuestos. Los resultados de los procedimientos de ataque se presentan y se describe la optimización de las técnicas para la fabricación de estructuras suspendidas de NCD y materiales III-N. En un capítulo posterior se estudia el crecimiento de AlN por pulverización catódica. Como se ha calculado en esta disertación para obtener una actuación eficiente de MEMS, las capas de AlN han de ser finas, típicamente d < 200 nm, lo que supone serias dificultades para la obtención de capas orientadas con respuesta piezoeléctrica. Las condiciones de depósito se han mapeado para identificar las fronteras que proporcionan el crecimiento de material orientado desde los primeros pasos del proceso. Además, durante la optimización de los procesos de ataque se estudió un procedimiento para fabricar películas de GaN nanoporoso. Estas capas porosas pueden servir como capas sacrificiales para la fabricación de estructuras suspendidas de GaN con baja tensión residual o como capas para mejorar la funcionalización superficial de sensores químicos o biológicos. El proceso de inducción de poros se discutirá y también se presentarán experimentos de ataque y funcionalización. En segundo lugar, se han determinado las propiedades mecánicas del NCD y de los materiales III-N. Se han fabricado varias estructuras suspendidas para la medición del módulo de Young y de la tensión residual. Además, las estructuras de NCD se midieron en resonancia para calcular el rendimiento de los dispositivos en términos de frecuencia y factor de calidad. Se identificaron los factores intrínsecos y extrínsecos que limitan ambas figuras de mérito y se han desarrollado modelos para considerar estas imperfecciones en las etapas de diseño de los dispositivos. Por otra parte, los materiales III-N normalmente presentan grandes gradientes de deformación residual que causan la deformación de las estructuras al ser liberadas. Se han medido y modelado estos efectos para los tres materiales binarios del sistema para proporcionar puntos de interpolación que permitan predecir las características de las aleaciones del sistema III-N. Por último, los datos recabados se han usado para desarrollar modelos analíticos y numéricos para el diseño de varios dispositivos. Se han estudiado las propiedades de transducción y se proporcionan topologías optimizadas. En el último capítulo de esta disertación se presentan diseños optimizados de los siguientes dispositivos: _ Traviesas y voladizos de AlN=NCD con actuación piezoeléctrica aplicados a nanoconmutadores de RF para señales de alta potencia. _ Membranas circulares de AlN=NCD con actuación piezoeléctrica aplicadas a lentes sintonizables. _ Filtros ópticos Fabry-Pérot basados en cavidades aéreas y membranas de GaN actuadas electrostáticamente. En resumen, se han desarrollado unos nuevos procedimientos optimizados para la fabricación de estructuras de NCD y materiales III-N. Estas técnicas se han usado para producir estructuras que llevaron a la determinación de las principales propiedades mecánicas y de los parámetros de los dispositivos necesarios para el diseño de MEMS. Finalmente, los datos obtenidos se han usado para el diseño optimizado de varios dispositivos demostradores. ABSTRACT Micro Electro Mechanical Systems (MEMS) have proven to be a successful family of devices that can be used as a platform for the development of devices with applications in optics, communications, signal processing and sensorics. Standard MEMS devices are usually fabricated using silicon based materials. However, the performance of these MEMS can be improved if other material systems are used. For instance, nanocrystalline diamond (NCD) offers excellent mechanical properties, optical transparency and ease of surface functionalization. On the other hand, the (In; Ga; Al)N material system, the III-N materials, can be used to produce single crystal structures with high mechanical and chemical sensitivity. Also, AlN can be deposited by reactive sputtering on various substrates, including NCD, to form oriented polycrystalline layers with high piezoelectric response. In addition, both NCD and III-N materials exhibit high thermal and chemical stability, which makes these material the perfect choice for the development of devices for high temperatures, harsh environments and even biocompatible applications. In this thesis these materials have been used for the design and measurement of technological demonstrators. Three main objectives have been pursued: _ Development of suitable fabrication processes. _ Measurement of the material mechanical properties and device performance limiting factors. _ Use the gathered data to design complex demonstrator devices. In a first part of the thesis several fabrication processes have been addressed. The stability of these materials hinders the etching of the layers and hampers the production of free standing structures. The first chapters of this dissertation are devoted to the development of a dry patterning etching process and to sacrificial etching optimization of several proposed substrates. The results of the etching processes are presented and the optimization of the technique for the manufacturing of NCD and III-N free standing structures is described. In a later chapter, sputtering growth of thin AlN layers is studied. As calculated in this dissertation, for efficient MEMS piezoelectric actuation the AlN layers have to be very thin, typically d < 200 nm, which poses serious difficulties to the production of c-axis oriented material with piezoelectric response. The deposition conditions have been mapped in order to identify the boundaries that give rise to the growth of c-axis oriented material from the first deposition stages. Additionally, during the etching optimization a procedure for fabricating nanoporous GaN layers was also studied. Such porous layers can serve as a sacrificial layer for the release of low stressed GaN devices or as a functionalization enhancement layer for chemical and biological sensors. The pore induction process will be discussed and etching and functionalization trials are presented. Secondly, the mechanical properties of NCD and III-N materials have been determined. Several free standing structures were fabricated for the measurement of the material Young’s modulus and residual stress. In addition, NCD structures were measured under resonance in order to calculate the device performance in terms of frequency and quality factor. Intrinsic and extrinsic limiting factors for both figures were identified and models have been developed in order to take into account these imperfections in the device design stages. On the other hand, III-N materials usually present large strain gradients that lead to device deformation after release. These effects have been measured and modeled for the three binary materials of the system in order to provide the interpolation points for predicting the behavior of the III-N alloys. Finally, the gathered data has been used for developing analytic and numeric models for the design of various devices. The transduction properties are studied and optimized topologies are provided. Optimized design of the following devices is presented at the last chapter of this dissertation: _ AlN=NCD piezoelectrically actuated beams applied to RF nanoswitches for large power signals. _ AlN=NCD piezoelectrically actuated circular membranes applied to tunable lenses. _ GaN based air gap tunable optical Fabry-Pérot filters with electrostatic actuation. On the whole, new optimized fabrication processes has been developed for the fabrication of NCD and III-N MEMS structures. These processing techniques was used to produce structures that led to the determination of the main mechanical properties and device parameters needed for MEMS design. Lastly, the gathered data was used for the design of various optimized demonstrator devices.

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 Å, enabling direct visualization of α-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i.e., insertion of peptides, ≈10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 Å resolution. Hepatitis B virus capsids are icosahedral particles, ≈300 Å in diameter, made up of T-shaped dimers (subunit Mr, 16–21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and −7 (in the residual propeptide) in the “e-antigen” form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N′-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)—both pharmacologic (with a clear dose–response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.