NADPH-CYTOCHROME P-450 REDUCTASE: EVIDENCE FOR TWO SEPARATE STRUCTURAL DOMAINS AND ISOLATION AND CHARACTERIZATION OF THE MEMBRANE ASSOCIATED SEGMENT

Homogenous detergent-solubilized NADPH-Cytochrome P-450 reductase was incorporated into microsomes and liposomes. This binding occurred spontaneously at temperatures between 4(DEGREES) and 37(DEGREES) and appeared to involve hydrophobic forces as the binding was not disrupted by 0.5 M sodium chloride. This exogenously-added reductase was active catalytically towards native cytochrome P-450, suggesting an association with the microsomal membrane similar to endogenous reductase. Homogeneous detergent-solubilized reductase was disaggregated by Renex-690 micelles, confirming the presence of a hydrophobic combining region on the enzyme. In contrast to these results, steapsin protease-solubilized reductase was incapable of microsomal attachment and did not interact with Renex-690 micelles. Detergent-solubilized reductase (76,500 daltons) was converted into a form with the electrophoretic mobility of steapsin protease-solubilized reductase (68,000 daltons) and a 12,500 dalton peptide (as determined by polyacrylamide-SDS gel electrophoresis) when the liposomal-incorporated enzyme was incubated with steapsin protease. The 68,000 dalton fragment thus obtained had properties identical with steapsin protease-solubilized reductase, i.e. it was catalytically active towards cytochrome c but inactive towards cytochrome P-450 and did not bind liposomes. The 12,500 dalton fragment remained associated with the liposomes when the digest was fractionated by gel filtration, suggesting that this is the segment of the enzyme which is embedded in the phospholipid bilayer. Thus, detergent-solubilized reductase appears to contain a soluble catalytic domain and a separate and separable membrane-binding domain. This latter domain is required for attaching the enzyme to the membrane and also to facilitate the catalytic interaction between the reductase and its native electron acceptor, cytochrome P-450. The membrane-binding segment of the reductase was isolated by preparative gel electrophoresis in SDS following its generation by proteolytic treatment of liposome-incorporated reductase. The peptide has a molecular weight of 6,400 as determined by gel filtration in 8 M guanidine hydrochloride and has an amino acid composition which is not especially hydrophobic. Following removal of SDS and dialysis out of 6 M urea, the membrane-binding peptide was unable to inhibit the activity of a reconstituted system containing purified reductase and cytochrome P-450. Moreover, when reductase and cytochrome P-450 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptide were used. Thus, the membrane-binding peptide was ineffective as an inhibitor of mixed function oxidase activity, suggesting perhaps that it facilitates catalysis by anchoring the catalytic domain of the reductase proximal to cytochrome P-450 (i.e. in the same mixed micelle) rather than through a specific interaction with cytochrome P-450. ^

Detecting the signature of natural selection with microsatellites

Natural selection is one of the major factors in the evolution of all organisms. Detecting the signature of natural selection has been a central theme in evolutionary genetics. With the availability of microsatellite data, it is of interest to study how natural selection can be detected with microsatellites. ^ The overall aim of this research is to detect signatures of natural selection with data on genetic variation at microsatellite loci. The null hypothesis to be tested is the neutral mutation theory of molecular evolution, which states that different alleles at a locus have equivalent effects on fitness. Currently used tests of this hypothesis based on data on genetic polymorphism in natural populations presume that mutations at the loci follow the infinite allele/site models (IAM, ISM), in the sense that at each site at most only one mutation event is recorded, and each mutation leads to an allele not seen before in the population. Microsatellite loci, which are abundant in the genome, do not obey these mutation models, since the new alleles at such loci can be created either by contraction or expansion of tandem repeat sizes of core motifs. Since the current genome map is mainly composed of microsatellite loci and this class of loci is still most commonly studied in the context of human genome diversity, this research explores how the current test procedures for testing the neutral mutation hypothesis should be modified to take into account a generalized model of forward-backward stepwise mutations. In addition, recent literature also suggested that past demographic history of populations, presence of population substructure, and varying rates of mutations across loci all have confounding effects for detecting signatures of natural selection. ^ The effects of the stepwise mutation model and other confounding factors on detecting signature of natural selection are the main results of the research. ^

Splendid Isolation

K. B.

Isolation and characterization of cDNAs encoding novel homeoproteins from chondrocytes

Bone morphogenesis is a complex biological process. The multistep process of chondrogenesis is the most important aspect of endochondral bone formation. To study the mechanisms which control this multistep pathway of chondrogenesis during embryonic development, I started by isolating cDNAs encoding novel transcriptional factors from chondrocytes. Several such cDNAs encoding putative homeoproteins were identified from a rat chondrosarcoma cDNA preparation. I have been concentrating on characterizing two of these cDNAs. The deduced amino acid sequence of the first homeoprotein, Cart-1, contains a prd-type homeodomain. Northern hybridization and RNase protection analysis revealed that Cart-1 RNAs were present at high levels in a well differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 transcripts were also detected in primary chondrocytes, but not in numerous other cell types except very low levels in testis. In situ hybridization of rat embryos at different stages of development revealed relatively high levels of Cart-1 RNAs in prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. It is speculated that Cart-1 might play an important role in chondrogenesis. The second putative homeoprotein, rDlx, contains a Distal-less-like homeodomain. rDlx RNAs were also present at high levels in the rat chondrosarcoma tumor and in the cell line derived from this tumor. In situ hybridization of rat embryos revealed high levels of rDlx transcripts in the developing cartilages and perichondria of mature cartilages. rDlx transcripts were also detected in a number of nonchondrogenic tissues such as forebrain, otic vesicles, olfactory epithelia, apical ectodermal ridge (AER) of limb buds, the presumptive Auerbach ganglia of gastrointestinal tract. The unique expression pattern of rDlx suggests that it might play important roles in chondrogenesis and other aspects of embryogenesis. ^

Isolation and characterization of revertant cell lines expressing oncogenic rat {\it neu\/}

The neu gene encodes the transmembrane tyrosine kinase growth factor receptor, p185. To study neu induced cellular transformation, we developed revertant cells from the neu transformed NIH 3T3 cell line, B104-1-1, by treating the cells with the chemical mutagen ethylmethane sulfonate. The morphologically normal revertant cells were first selected by their ability to either attach to culture plates or survive in the presence of the cytotoxic reagents colchicine or 5-fluoro-2deoxyuridine. Two of the 21 candidate revertant cell lines isolated were further characterized and were found to lose their anchorage independence and ability to grow in 1% calf serum, indicating that they were nontransformed even though they still expressed p185 oncoprotein. The tyrosine residues of p185 in these two revertants were underphosphorylated, which may have contributed to their nontransformed status. Also, the p185 oncoprotein lacked significant tyrosine kinase activity. In addition, these revertants also resisted transformation by neu and several additional oncogenes (H-ras, N-ras, v-mos, v-abl, and v-fos) as determined by focus forming assays. These results indicated that we had successfully developed, from neu transformed cells, revertants which exhibited defective tyrosine phosphorylation and kinase activity of the neu oncoprotein. The results also suggested that neu and several other oncogenes may share common elements in their pathways for the induction of cellular transformation. ^

Isolation and characterization of a novel human Mix -like homeobox gene MIXL

Molecular events involved in specification of early hematopoietic system are not well known. In Xenopus, a paired-box homeodomain family (Mix.1–4) has been implicated in this process. Although Mix-like homeobox genes have been isolated from zebrafish (bon), chicken (CMIX) and mice (MmI/MIXL1), isolation of a human Mix-like gene has remained elusive. ^ We have recently isolated and characterized a novel human Mix-like homeobox gene with a predicted open reading frame of 232 amino acids designated the Mix.1 homeobox (Xenopus laevis)-like gene (MIXL). The overall identity of this novel protein to CMIX and MmI/MIXL1 is 41% and 69%, respectively. However, the identity in the homeodomain is 66% to that of Xenopus Mix.1, 79% to that of CMIX, and 94% to that of MmI/MIXL1. In normal hematopoiesis, MIXL expression appears to be restricted immature B and T lymphoid cells. Several acute leukemic cell lines of B, T and myeloid lineages express MIXL suggesting a survival/block in differentiation advantage. Furthermore, Xenopus animal cap assay revealed that MIXL could induce expression of the α-globin gene, suggesting a functional conservation of the homeodomain. ^ Biochemical analysis revealed that MIXL proteins are phosphorylated at multiple sites. Immunoprecipitation and immunoblotting confirmed that MIXL is tyrosine phosphorylated. Mutational analysis determined that Tyr20 appears to be the site for phosphorylation. However, deletion analysis preliminarily showed that the proline-rich domain appears not to be necessary for tyrosine phosphorylation. The novel finding will help us make a deeper understanding of the regulation on homeodomain proteins by rarely reported tyrosine phosphorylation. ^ Taken together, isolation of the MIXL gene is the first step toward understanding novel regulatory circuits in early hematopoietic differentiation and malignant transformation. ^

Upwelling and isolation in oxygen-depleted anticyclonic modewater eddies and implications for nitrate cycling

The physical (temperature, salinity, velocity) and biogeochemical (oxygen, nitrate) structure of an oxygen depleted coherent, baroclinic, anticyclonic mode-water eddy (ACME) is investigated using high-resolution autonomous glider and ship data. A distinct core with a diameter of about 70 km is found in the eddy, extending from about 60 to 200 m depth and. The core is occupied by fresh and cold water with low oxygen and high nitrate concentrations, and bordered by local maxima in buoyancy frequency. Velocity and property gradient sections show vertical layering at the flanks and underneath the eddy characteristic for vertical propagation (to several hundred-meters depth) of near inertial internal waves (NIW) and confirmed by direct current measurements. A narrow region exists at the outer edge of the eddy where NIW can propagate downward. NIW phase speed and mean flow are of similar magnitude and critical layer formation is expected to occur. An asymmetry in the NIW pattern is seen that possible relates to the large-scale Ekman transport interacting with ACME dynamics. NIW/mean flow induced mixing occurs close to the euphotic zone/mixed layer and upward nutrient flux is expected and supported by the observations. Combing high resolution nitrate (NO3-) data with the apparent oxygen utilization (AOU) reveals AOU:NO3- ratios of 16 which are much higher than in the surrounding waters (8.1). A maximum NO3- deficit of 4 to 6 µmol kg-1 is estimated for the low oxygen core. Denitrification would be a possible explanation. This study provides evidence that the recycling of NO3-, extracted from the eddy core and replenished into the core via the particle export, may quantitatively be more important. In this case, the particulate phase is of keys importance in decoupling the nitrogen from the oxygen cycling.

Detection and Isolation of Simultaneous Additive and Parametric Faults in Nonlinear Stochastic Dynamical Systems

This paper presents a new fault detection and isolation scheme for dealing with simultaneous additive and parametric faults. The new design integrates a system for additive fault detection based on Castillo and Zufiria, 2009 and a new parametric fault detection and isolation scheme inspired in Munz and Zufiria, 2008 . It is shown that the so far existing schemes do not behave correctly when both additive and parametric faults occur simultaneously; to solve the problem a new integrated scheme is proposed. Computer simulation results are presented to confirm the theoretical studies.

Fault detection and isolation on a noisy nonlinear circuit

In this paper fault detection and isolation (FDI) schemes are applied in the context of the surveillance of emerging faults in an electrical circuit. The FDI problem is studied on a noisy nonlinear circuit, where both abrupt and incipient faults in the voltage source are considered. A rigorous analysis of fault detectability precedes the application of the fault detection (FD) scheme; then, the fault isolation (FI) phase is accomplished with two alternative FI approaches, proposed as new extensions of that FD approach. Numerical simulations illustrate the applicability of the mentioned schemes.

Fault isolation schemes for a class of continuous-time stochastic dynamical systems

In this paper a new method for fault isolation in a class of continuous-time stochastic dynamical systems is proposed. The method is framed in the context of model-based analytical redundancy, consisting in the generation of a residual signal by means of a diagnostic observer, for its posterior analysis. Once a fault has been detected, and assuming some basic a priori knowledge about the set of possible failures in the plant, the isolation task is then formulated as a type of on-line statistical classification problem. The proposed isolation scheme employs in parallel different hypotheses tests on a statistic of the residual signal, one test for each possible fault. This isolation method is characterized by deriving for the unidimensional case, a sufficient isolability condition as well as an upperbound of the probability of missed isolation. Simulation examples illustrate the applicability of the proposed scheme.

High isolation series-shunt photoconductive microwave switch

This article presents a wide band compact high isolation photoconductive switch, which is based on the series-shunt switch design with three photoconductive switches made of diced high-resistivity silicon wafer placed over a microstrip gap and activated by 808-nm near-infrared laser diodes. The switch shows an insertion loss of 1.2 dB and an isolation of 44.8 dB at 2 GHz. It is easy to operate and control by light, high-speed, electromagnetically transparent and it does not require any biasing circuits.

Finite element code-based modeling of a multi-feature isolation system and passive alleviation of possible inner pounding

The existing seismic isolation systems are based on well-known and accepted physical principles, but they are still having some functional drawbacks. As an attempt of improvement, the Roll-N-Cage (RNC) isolator has been recently proposed. It is designed to achieve a balance in controlling isolator displacement demands and structural accelerations. It provides in a single unit all the necessary functions of vertical rigid support, horizontal flexibility with enhanced stability, resistance to low service loads and minor vibration, and hysteretic energy dissipation characteristics. It is characterized by two unique features that are a self-braking (buffer) and a self-recentering mechanism. This paper presents an advanced representation of the main and unique features of the RNC isolator using an available finite element code called SAP2000. The validity of the obtained SAP2000 model is then checked using experimental, numerical and analytical results. Then, the paper investigates the merits and demerits of activating the built-in buffer mechanism on both structural pounding mitigation and isolation efficiency. The paper addresses the problem of passive alleviation of possible inner pounding within the RNC isolator, which may arise due to the activation of its self-braking mechanism under sever excitations such as near-fault earthquakes. The results show that the obtained finite element code-based model can closely match and accurately predict the overall behavior of the RNC isolator with effectively small errors. Moreover, the inherent buffer mechanism of the RNC isolator could mitigate or even eliminate direct structure-tostructure pounding under severe excitation considering limited septation gaps between adjacent structures. In addition, the increase of inherent hysteretic damping of the RNC isolator can efficiently limit its peak displacement together with the severity of the possibly developed inner pounding and, therefore, alleviate or even eliminate the possibly arising negative effects of the buffer mechanism on the overall RNC-isolated structural responses.

Memory isolation in many-core embedded systems

The current approach to developing mixed-criticality sys- tems is by partitioning the hardware resources (processors, memory and I/O devices) among the different applications. Partitions are isolated from each other both in the temporal and the spatial domain, so that low-criticality applications cannot compromise other applications with a higher level of criticality in case of misbehaviour. New architectures based on many-core processors open the way to highly parallel systems in which each partition can be allocated to a set of dedicated proces- sor cores, thus simplifying partition scheduling and temporal separation. Moreover, spatial isolation can also benefit from many-core architectures, by using simpler hardware mechanisms to protect the address spaces of different applications. This paper describes an architecture for many- core embedded partitioned systems, together with some implementation advice for spatial isolation.

Análisis de sistemas eléctricos en microsatélites. Desarrollo de módulos para una Concurrent Design Facility (CFD)para el desarrollo y análisis de misiones espaciales = Analysis of microsatellites power subsystems (battery testing). Development and programming of a new Propulsion Module for a space mission Concurrent Design Facility (CDF)

El objetivo de este proyecto es recoger y explicar el conjunto de tareas realizadas durante el proceso de colaboración llevado a cabo en el Instituto de Microgravedad “Ignacio Da Riva” durante el curso académico 2014/2015, las cuales han conformado las prácticas externas cursadas en la titulación de Grado en Ingeniería Aeroespacial, y el Trabajo de Fin de Grado de la misma titulación. En este documento se pretende,además, poner de manifiesto la rigurosidad con la que se trabaja en el ámbito espacial y la importancia de los protocolos y procedimientos para asegurar un resultado adecuado en los trabajos realizados. Una parte importante del proyecto detalla los procesos de monitorización y mantenimiento de la batería del satélite universitario UPMSat-2 cuyo lanzamiento está previsto para el año próximo y el cual será el segundo satélite del mundo en incorporar una batería de Ión-Litio.