Running-In of Metal-to-Metal Seals and its Influence on Sealing Ability:With application to the design of biodegradable thread compounds

Metal-to-metal sealing of casing connections is affected by running-in because it determines the topography of the gap between the contacting surfaces by wear and plastic deformation during assembly under the influence of the thread compound, coatings and the initial surface topography. The work in this thesis concerns the mechanisms related to these elements of the tribosystem and how they affect running-in of the metal-to-metal seal tribosystem and ultimately influence the sealing ability.The research was driven by a need to reduce costs in particular of the qualification of premium connections. Furthermore, increased understanding of the barriers in the well is important for well engineering. Understanding was needed on galling during assembly which is an often occurring failure mechanism as well as the protective mechanisms behind the applied coatings. In addition, the Oslo-Paris Convention for the protection of the Marine Environment of the North-East Atlantic (OSPAR) demands future substitution of mineral oils and the (heavy) metal additives used in the American Petroleum Institute (API) modified thread compound by biodegradable alternatives. To this end a combined experimental and modelling approach was applied.The existing thread compounds were shown, with pin-on-disc, anvil-on-strip and Shell Sealing Mock-Up Rig (SSMUR) tests, to provide relatively minor protection to initiation of galling in uncoated contacts. This was shown to be because of squeeze out of the formed tribofilms, limited adsorption of the additives and the lack of replenishment by the plan parallel contact configuration coming from the turned surface topography. These mechanisms only added 60mm of additional sliding length, before failure, with API modified thread compound compared to a plain mineral oil.In relation to the substitution of API modified for biodegradable alternatives, the elevated temperature degradation mechanisms of (environmentally acceptable) thread compounds were studied using Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), high temperature rheometry and pin-on-disc. Thread compounds were shown to fail because of evaporation and oxidation leading to starved lubrication conditions. The system subsequently enters a severe adhesive wear regime sometimes exacerbated by abrasive action of the hard metal oxide particles present in environmentally acceptable compounds. The found failure mechanisms and the developed test protocol were validated and successfully mitigated by the development of a prototype thread compound.The presence of phosphate conversion coatings proved to be a dominating factor in the running-in of the metal-to-metal seal. This was shown to be caused by two main mechanisms using various tribological experiments and analytical techniques. On the uncoated counter surface a durable tribofilm was formed by physical adsorption of phosphate debris particles through a shear stress activation mechanism. From the same debris particles a smooth glaze layer was generated on the phosphated surface which possesses a substantial hardness after dry sliding. However, this hardness was much lower after lubricated sliding and was shown to be related to the particle size which generated the glaze layer. The combination of these mechanisms resulted in a wear process that could satisfactorily be described by the energy dissipated in the sliding contact.Finally, it was shown with experimental data and a simple running-in model that the combination of plastic deformation of the waviness of the turned surface topography and wear of the phosphate coating determine the running-in behaviour. It was found that the surface runs-in within 40mm sliding length after which the wear regime transitioned to mild wear. The combination of severe initial wear by plastic deformation of the waviness and the generation of a smooth glaze layer created a conformal sealing configuration with multiple concentrated line contacts along the circumference. This created the most robust sealing configuration compared to configurations that did not have a distinct lay.<br/

Hypoxic stress suppresses RNA polymerase III recruitment and tRNA gene transcription in cardiomyocytes

RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes

Mutation of RNA Pol III Subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and Disrupts Zebrafish Digestive Development

The role of RNA polymerase III (Pol III) in developing vertebrates has not been examined. Here, we identify a causative mutation of the second largest Pol III subunit, polr3b, that disrupts digestive organ development in zebrafish slim jim (slj) mutants. The slj mutation is a splice-site substitution that causes deletion of a conserved tract of 41 amino acids in the Polr3b protein. Structural considerations predict that the slj Pol3rb deletion might impair its interaction with Polr3k, the ortholog of an essential yeast Pol III subunit, Rpc11, which promotes RNA cleavage and Pol III recycling. We engineered Schizosaccharomyces pombe to carry an Rpc2 deletion comparable to the slj mutation and found that the Pol III recovered from this rpc2-Δ yeast had markedly reduced levels of Rpc11p. Remarkably, overexpression of cDNA encoding the zebrafish rpc11 ortholog, polr3k, rescued the exocrine defects in slj mutants, indicating that the slj phenotype is due to deficiency of Rpc11. These data show that functional interactions between Pol III subunits have been conserved during eukaryotic evolution and support the utility of zebrafish as a model vertebrate for analysis of Pol III function

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

Background: Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.[br/] Results: The best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.[br/] Conclusions: RNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery

Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro

Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30–50 % at a concentration of 1 μM. Osteoclasts were found to express the A2A, A2B and A3 receptors; however, neither adenosine (≤100 μM) nor 2-chloroadenosine (≤10 μM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone

NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival.

Neutrophil lifespan is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis related genes, the most highly regulated of these being NR4A2 and NR4A3 Direct PKA activation by the site-selective PKA agonist pair N6/8-AHA and treatment with endogenous activators of PKA, including adenosine and PGE2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and siRNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologues in zebrafish larvae significantly reduces absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis

Purinergic signalling and immune cells

This review article provides a historical perspective on the role of purinergic signalling in the regulation of various subsets of immune cells from early discoveries to current understanding. It is now recognised that adenosine 5'-triphosphate (ATP) and other nucleotides are released from cells following stress or injury. They can act on virtually all subsets of immune cells through a spectrum of P2X ligand-gated ion channels and G protein-coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and adenosine exerts additional regulatory effects through its own receptors. The resulting effect ranges from stimulation to tolerance depending on the amount and time courses of nucleotides released, and the balance between ATP and adenosine. This review identifies the various receptors involved in the different subsets of immune cells and their effects on the function of these cells

The Late Pleistocene and Holocene Evolution of The North Caspian Plain

The rapid rates of sea level change in the Caspian Sea since its formation (Proterozoic) have resulted in unique sedimentary sequences, including many oil and gas reservoirs. An important hydrocarbon province is the fine grained-sandstone of the Productive Series in the South Caspian Basin that consist for at least some part of sheetflood sandstones. Their sedimentology and reservoir architecture remain uncertain. This study reconstructs the chrono-stratigraphic framework of the low-gradient North-East Caspian plain between the Ural and Emba Rivers, and its potential as a reservoir analogue for Productive Series. Because of the similarities between the sedimentary systems, like rapid sea level changes and the absence of a shelf break. The data for this study consist of 26 shallow core- and 10 outcrop-descriptions and include the analyses of selected samples for Optically Stimulated Luminescence dating, radiocarbon dating and grain-size distribution analyses. Three sea-level cycles (I-III) were identified depositing four different sedimentary sequences (a-d): (I-a) marine clays (41 ka BP) followed by a regression resulting in an erosional surface, (II-b) transgressive shallow marine silty clays and sandy barrier deposits (16-17 ka BP) (locally identified as ‘Khvalynian chocolate clays’), (II-c) Terminal Splay Complex consisting of clayey sands (9-11 ka BP) related to a major regressive event, that are eroded into a deflation landscape (e.g. yardangs), locally known as ‘Baer-hills’ and Sors’ and (III-d) present day inter-yardang deposits lagoonal- and playa- like deposits consisting of fine silty clays. Preservation of these fines is closely related to sea-level oscillations. Possibly a fourth sea-level cycle occurred before 41 ka BP depositing marine clays. The identification of a Holocene terminal splay complex and yardang-formation gives new insight in the regional sedimentary processes. More importantly this complex could serve as a new analogue for the Productive Series, giving alternative insights in reservoir architecture and internal connectivity, e.g. the Productive Series could be more terrestrial of origin despite the absence of evidence for sub-aerial exposure.Applied GeologyApplied Earth SciencesCivil Engineering and Geoscience

Finite Element Methods with exact geometry representation: IsoGeometric Analysis, NURBS Enhanced Finite Element Method and AnisoGeometric Analysis

Traditionally, geometry has been represented differently in the field of Computer Aided Design (CAD) and Finite Element Analysis (FEA). This means that the CAD geometry, which can be seen as exact, must be converted to an Analysis Suitable Geometry (ASG) for input in a FEA program. A cumbersome and time consuming process, more commonly known as meshing. Furthermore, most engineering analysis techniques use linear or quadratic approximations of the originally exact geometry. Besides the geometry error, these crude geometry approximations can give rise to numerical errors such as spurious oscillations. In order to avoid these problems an integrated approach is necessary which unifies CAD and FEA.Aerospace Engineerin