Thermal profile of a near-adiabatic compression process in a cylindrical tube and establishment of critical control elements for repeatable process control

The compressed gas industry and government agencies worldwide utilize "adiabatic compression" testing for qualifying high-pressure valves, regulators, and other related flow control equipment for gaseous oxygen service. This test methodology is known by various terms including adiabatic compression testing, gaseous fluid impact testing, pneumatic impact testing, and BAM testing as the most common terms. The test methodology will be described in greater detail throughout this document but in summary it consists of pressurizing a test article (valve, regulator, etc.) with gaseous oxygen within 15 to 20 milliseconds (ms). Because the driven gas1 and the driving gas2 are rapidly compressed to the final test pressure at the inlet of the test article, they are rapidly heated by the sudden increase in pressure to sufficient temperatures (thermal energies) to sometimes result in ignition of the nonmetallic materials (seals and seats) used within the test article. In general, the more rapid the compression process the more "adiabatic" the pressure surge is presumed to be and the more like an isentropic process the pressure surge has been argued to simulate. Generally speaking, adiabatic compression is widely considered the most efficient ignition mechanism for directly kindling a nonmetallic material in gaseous oxygen and has been implicated in many fire investigations. Because of the ease of ignition of many nonmetallic materials by this heating mechanism, many industry standards prescribe this testing. However, the results between various laboratories conducting the testing have not always been consistent. Research into the test method indicated that the thermal profile achieved (i.e., temperature/time history of the gas) during adiabatic compression testing as required by the prevailing industry standards has not been fully modeled or empirically verified, although attempts have been made. This research evaluated the following questions: 1) Can the rapid compression process required by the industry standards be thermodynamically and fluid dynamically modeled so that predictions of the thermal profiles be made, 2) Can the thermal profiles produced by the rapid compression process be measured in order to validate the thermodynamic and fluid dynamic models; and, estimate the severity of the test, and, 3) Can controlling parameters be recommended so that new guidelines may be established for the industry standards to resolve inconsistencies between various test laboratories conducting tests according to the present standards?

The functional and structural interactions between v-{\it mos\/} proteins and cell cycle control elements

The v-mos gene of Moloney murine sarcoma virus (Mo-MuSv) encodes a serine/threonine protein kinase capable of inducing cellular transformation. The c-mos protein is an important cell cycle regulator that functions during meiotic cell division cycles in germ cells. The overall function of c-mos in controlling meiosis is becoming better understood but the role of v-mos in malignant transformation of cells is largely unknown.^ In this study, v-mos protein was shown to be phosphorylated by M phase kinase in vitro and in vivo. The kinase activity and neoplastic transforming ability of v-mos is positively regulated by the phosphorylation. Together with the earlier finding of activation of M phase kinase by c-mos, these results raise the possibility of mutual regulation between M phase kinase and mos kinases.^ In addition to its functional interaction with the M phase kinase, the v-mos protein was shown to be present in the same protein complex with a cyclin-dependent kinase (cdk). In addition, an antibody that recognizes the cdk proteins was shown to co-precipitate the v-mos proteins in the interphase and mitotic cells transformed by p85$\sp{\rm gag-mos}$. Cdk proteins have been shown to be associated with nonmitotic cyclins which are potential oncogenes. The perturbation of cdk kinase or the activation of non-mitotic cyclins as oncogenes by v-mos could contribute directly to v-mos induced cellular transformation. v-mos proteins were also shown to interact with tubulin and vimentin, the essential components of microtubules and type IV intermediate filaments, respectively. The organizations of both microtubules and intermediate filaments are cell cycle-regulated. These results suggest that the v-mos kinase could be directly involved in inducing morphological changes typically seen in transformed cells.^ The interactions between the v-mos protein and these cell cycle control elements in regards to v-mos induced neoplastic transformation are discussed in detail in the text. ^

The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia.

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia.

Analysen der beiden cis-regulierenden Sequenzelemente translational control element (TCE) und cytoplasmic polyadenylation element (CPE) in der Drosophila-Spermatogenese

Ein essentieller Bestandteil in dem Mechanismus der Translationskontrolle sind RNA-Pro­tein-Wechselwirkungen. Solche Interaktionen konnten in Translationssystemen an zwei unabhängigen cis-regulierenden Elementen durch in vitro-Bindungsanalysen mit individu­ellen rekombinanten Proteinen dokumentiert werden. Im Fall des translational control elements (TCE), welches ein konserviertes Sequenz-Ele­ment in der Mst(3)CGP-Genfamilie darstellt, wird eine negative Translationskontrolle durch die Bindung der Proteine CG3213, CG12470, CG1898, dFMR1, Exuperantia und Orb2 an diese Sequenz vermittelt (Stinski, 2011). Neben den in Bindungsstudien positiv getesteten Kandidaten dFMR1 und Orb2 (Stinski, 2011) wurde in der vorliegenden Dis­sertation CG3213 als weiterer direkter Bindungspartner an das TCE dokumentiert. Ein Abgleich der genomweiten Zusammenstellung von Proteininteraktionen in der Datenbank InterologFinder lieferte zwei weitere potentielle Kandidaten: CG34404 und CG3727. Al­lerdings schließen Northern-Analysen und das Proteinexpressionsmuster eine zentrale Rolle in der Drosophila-Spermatogenese für diese nahezu aus. In Kolokalisationsstudien einiger TCE-Komplex-Kandidaten mit CG3213 als Referenz konnten eindeutige Überein­stimmungen der Fluoreszenzmuster mit CG12470 in der postmeiotischen Phase be­schrieben werden, wohingegen mit Orb2 (postmeiotisch) und CG1898 (prämeiotisch) nur eine geringe Kolokalisation erkannt wurde. Punktstrukturen in den Verteilungsmustern sowohl von CG3213 als auch von CG12470 ließen sich nicht mit ER- und mitochondrien­spezifischen Markern korrelieren. Im Anschluss der Meiose konnte eine deutliche Intensitätserhöhung des CG3213-Proteins beobachtet werden, was eventuell durch eine veränderte Translationseffizienz zustande kommen könnte. Exuperantia (Exu) stellt einen bekannten Regulator für eine Reihe von translationskontrollierten mRNAs dar (Wang und Hazelrigg, 1994). Die Quantifizierungen der CG3213-mRNA in exu-mutantem Hintergrund bestätigen, dass auch die Transkript­menge der CG3213-mRNA durch Exu reguliert wird, was die obige Interpretation stützen würde. Für das zweite cis-regulierende Element, das cytoplasmic polyadenylation element (CPE), konnte eine direkte Bindung mit dem CPEB-Homolog in Drosophila (Orb2) gezeigt wer­den, welches auch eine Komponente des mst87F-RNP-Komplexes ist. Ein vermuteter Interaktionspartner dieses CPEBs ist Tob, weshalb die Verteilung beider Proteine in einem Kombinationsstamm verglichen wurde. In dem teilweise übereinstimmenden Fluoreszenz­muster ist Tob an den distalen Spermatidenenden auffallend konzentriert. Das gesamte Tob-Muster jedoch legt eine Verteilung in den Mitochondrien nahe, wie die MitoTracker®-Färbung belegt. Somit wurde erstmals ein Mitglied der Tob/BTG-Genfamilie in der Droso­phila-Spermatogenese mit Mitochondrien in Verbindung gebracht. Die Lokalisierung die­ser Proteine ist bislang unklar, jedoch konnte eine Kernlokalisation trotz der N-terminalen NLS-Sequenz mit Hilfe einer Kernfärbung ausgeschlossen werden.

Doxycycline control of prion protein transgene expression modulates prion disease in mice

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547–5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at ≈50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.

Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions flanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts

The chloroplast gene rbcL encodes the large subunit of the CO2-fixing enzyme ribulose-bisphosphate carboxylase. In previous work a target for photo-accelerated degradation of Chlamydomonas reinhardtii rbcL transcripts in vivo was found to lie within the first 63 nucleotides, and a sequence element required for increasing the longevity of transcripts of rbcL-reporter genes was found to occur between nucleotides 170 and 350. Photo-accelerated degradation of rbcL transcripts has been found to require nucleotides 21 to 41. Transcript nucleotides lying between 329 and 334 and between 14 and 27 are essential for stabilizing transcripts in vivo; mutations in either region reduce the longevity of transcripts. It is postulated that the effectiveness of photo-accelerated endonuclease attacks on the nucleotide 21 to 41 region is reduced by physical blockage or distortion of the target sequence by interacting proteins that associate with nucleotides in the 14 to 27 and 329 to 334 regions of the transcripts. Both the nucleotide +329 to +334 stabilizing sequence of rbcL and a transcription enhancing sequence that lies between +126 and +170 encode well conserved (cyanobacteria through angiosperms) amino acid sequences; the evolution of expression control elements within the protein coding sequence of rbcL is considered.

Voltage control on an uninhabited autonomous vehicle electrical distribution system

More-electric vehicle technology is becoming prevalent in a number of transportation systems because of its ability to improve efficiency and reduce costs. This paper examines the specific case of an Uninhabited Autonomous Vehicle (UAV), and the system topology and control elements required to achieve adequate dc distribution voltage bus regulation. Voltage control methods are investigated and a droop control scheme is implemented on the system. Simulation results are also presented.

An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed.

Cell-targeted regulation of gene expression through synthetic RNA devices

The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function.

We describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation.

We next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein.

The foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.

A custom-built insulin resistance gene chip

Objectives/Aim—Microarray (gene chip) technology offers a powerful new tool for analyzing the expression of large numbers of genes in many experimental samples. The aim of this study was to design, construct, and use a gene chip to measure the expression levels of key genes in metabolic pathways related to insulin resistance.
Methods—We selected genes that were implicated in the development of insulin resistance, including genes involved in insulin signaling; glucose uptake, oxidation, and storage; fat uptake, oxidation, and storage; cytoskeletal components; and transcription factors. The key regulatory genes in the pathways were identified, along with other recently identified candidate genes such as calpain-10. A total of 242 selected genes (including 32 internal control elements) were sequence-verified, purified, and arrayed on aldehyde-coated slides.
Results—Where more than 1 clone containing the gene of interest was available, we chose those containing the genes in the 5' orientation and an insert size of around 1.5 kb. Of the 262 clones purchased, 56 (21%) were found to contain sequences other than those expected. In addition, 2 (1%) did not grow under standard conditions and were assumed to be nonviable. In these cases, alternate clones containing the gene of interest were chosen as described above. The current version of the Insulin Resistance Gene Chip contains 210 genes of interest, plus 48 control elements. A full list of the genes is available at http://www.hbs.deakin.edu.au/mru/research/gene_chip_tech/genechip_three.htm/.
Conclusions—The human Insulin Resistance Gene Chip that we have constructed will be a very useful tool for investigating variation in the expression of genes relevant to insulin resistance under various experimental conditions. Initially, the gene chip will be used in studies such as exercise interventions, fasting, euglycemic-hyperinsulinemic clamps, and administration of antidiabetic agents

Fototriangulação em bloco de imagens orbitais com modelos rigorosos baseados em pontos e retas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Retificador trifásico de 18 pulsos com estágio CC controlado por histerese constante

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

Abstract Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

Inducible endothelial cell-specific gene expression in transgenic mouse embryos and adult mice

Utilizing both the TET-OFF and TET-ON systems in combination with transcriptional control elements of the Tie-2 gene, we have established a series of transgenic activator and responder mice for TET-regulated endothelial cell-specific transgene expression in double transgenic mouse embryos and in adult mice. TET-regulated expression of LacZ reporter genes could be achieved in virtually all endothelia in mid gestation stage mouse embryos. In contrast in adult mice, using the very same Tie-2 tTA activator mouse strain, we observed striking differences of TET-induced gene expression from various inducible expression constructs in different vascular beds. Non-endothelial expression was never detected. The prominent differences in completeness of TET-induced endothelial expression highlight the still underestimated critical role of the responder mouse lines for uniform TET-induced gene expression in heterogeneous cell populations such as endothelial cells. Interestingly, in double transgenic mice inducibly expressing several different adhesion molecules, no adverse effects were observed even though these proteins were robustly expressed on endothelial cells in adult tissues. These transgenic model systems provide versatile tools for the TET-regulated manipulation of endothelial cell-specific gene expression in the entire embryonic vasculature and distinct vascular beds in adult mice.