A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

Inter-individual variability in optimal current direction for transcranial magnetic stimulation of the motor cortex

We evaluated inter-individual variability in optimal current direction for biphasic transcranial magnetic stimulation (TMS) of the motor cortex. Motor threshold for first dorsal interosseus was detected visually at eight coil orientations in 45° increments. Each participant (n = 13) completed two experimental sessions. One participant with low test–retest correlation (Pearson's r < 0.5) was excluded. In four subjects, visual detection of motor threshold was compared to EMG detection; motor thresholds were very similar and highly correlated (0.94–0.99). Similar with previous studies, stimulation in the majority of participants was most effective when the first current pulse flowed towards postero-lateral in the brain. However, in four participants, the optimal coil orientation deviated from this pattern. A principal component analysis using all eight orientations suggests that in our sample the optimal orientation of current direction was normally distributed around the postero-lateral orientation with a range of 63° (S.D. = 13.70°). Whenever the intensity of stimulation at the target site is calculated as a percentage from the motor threshold, in order to minimize intensity and side-effects it may be worthwhile to check whether rotating the coil 45° from the traditional posterior–lateral orientation decreases motor threshold.

Improved damage detectability in a wind turbine blade by optimal selection of vibration signal correlation coefficients

Piotr Omenzetter and Simon Hoell’s work within the Lloyd’s Register Foundation Centre for Safety and Reliability Engineering at the University of Aberdeen is supported by Lloyd’s Register Foundation. The Foundation helps to protect life and property by supporting engineering-related education, public engagement and the application of research.

THE HERPESVIRUS NUCLEAR EGRESS COMPLEX COMPONENT, UL31, IS RECRUITED TO SITES OF DNA DAMAGE THROUGH POLY(ADP-RIBOSE) BINDING

The herpes simplex virus (HSV) UL31 gene encodes a conserved member of the herpesvirus nuclear egress complex that not only functions in the egress of DNA-containing capsids from the nucleus, but is also required for optimal viral genome expression, replication and packaging into capsids. Here, we report that the UL31 protein from HSV-2 and the orthologous protein, ORF69, from Kaposi's sarcoma-associated herpesvirus (KSHV) are recruited to sites of DNA damage. Recruitment of UL31 to sites of DNA damage occurred in HSV-2 infected cells, but did not require other viral proteins. The N-terminus of UL31 contains sequences resembling a poly(ADP-ribose) (PAR) binding motif. As protein poly-ADP ribosylation (PARylation) is a hallmark of the DNA damage response we examined the relationship between PARylation and UL31 recruitment to DNA damage. While the PAR polymerase (PARP)1/2 inhibitor, olaparib, prevented UL31 recruitment to damaged DNA, KU55933 inhibition of signaling through the ataxia telangiectasia mutated (ATM) DNA damage response pathway had no effect. These findings were further supported by experiments demonstrating direct and specific interaction between HSV-2 UL31 and PAR using purified components. Co-transfection with the viral kinase Us3, known to phosphorylate UL31, inhibited UL31 recruitment to DNA damage but also prevented the recruitment of other proteins recruited to DNA damage sites. The viral E3 ubiquitin ligase ICP0 was observed to co-localize with UL31 in transfected cells in a manner that is independent of the PAR-binding ability of UL31. However, inhibition of PARP1/2/3 did not reduce the ability of HSV-2 to replicate and we observed reduced PAR levels in the nuclei of infected cells. This study reveals a previously unrecognized function for UL31 orthologs and may suggest that the recognition of PAR by UL31 is coupled to the nuclear egress of herpesvirus capsids, influences viral DNA replication and packaging, or possibly modulates the DNA damage response mounted by virally infected cells.

Cognitive Component Skills of Reading Comprehension in Developing Readers

Die Fähigkeit, geschriebene Texte zu verstehen, d.h. eine kohärente mentale Repräsentation von Textinhalten zu erstellen, ist eine notwendige Voraussetzung für eine erfolgreiche schulische und außerschulische Entwicklung. Es ist daher ein zentrales Anliegen des Bildungssystems Leseschwierigkeiten frühzeitig zu diagnostizieren und mithilfe zielgerichteter Interventionsprogramme zu fördern. Dies erfordert ein umfassendes Wissen über die kognitiven Teilprozesse, die dem Leseverstehen zugrunde liegen, ihre Zusammenhänge und ihre Entwicklung. Die vorliegende Dissertation soll zu einem umfassenden Verständnis über das Leseverstehen beitragen, indem sie eine Auswahl offener Fragestellungen experimentell untersucht. Studie 1 untersucht inwieweit phonologische Rekodier- und orthographische Dekodierfertigkeiten zum Satz- und Textverstehen beitragen und wie sich beide Fertigkeiten bei deutschen Grundschüler(inne)n von der 2. bis zur 4. Klasse entwickeln. Die Ergebnisse legen nahe, dass beide Fertigkeiten signifikante und eigenständige Beiträge zum Leseverstehen leisten und dass sich ihr relativer Beitrag über die Klassenstufen hinweg nicht verändert. Darüber hinaus zeigt sich, dass bereits deutsche Zweitklässler(innen) den Großteil geschriebener Wörter in altersgerechten Texten über orthographische Vergleichsprozesse erkennen. Nichtsdestotrotz nutzen deutsche Grundschulkinder offenbar kontinuierlich phonologische Informationen, um die visuelle Worterkennung zu optimieren. Studie 2 erweitert die bisherige empirische Forschung zu einem der bekanntesten Modelle des Leseverstehens—der Simple View of Reading (SVR, Gough & Tunmer, 1986). Die Studie überprüft die SVR (Reading comprehension = Decoding x Comprehension) mithilfe optimierter und methodisch stringenter Maße der Modellkonstituenten und überprüft ihre Generalisierbarkeit für deutsche Dritt- und Viertklässler(innen). Studie 2 zeigt, dass die SVR einer methodisch stringenten Überprüfung nicht standhält und nicht ohne Weiteres auf deutsche Dritt- und Viertklässler(innen) generalisiert werden kann. Es wurden nur schwache Belege für eine multiplikative Verknüpfung von Dekodier- (D) und Hörverstehensfertigkeiten (C) gefunden. Der Umstand, dass ein beachtlicher Teil der Varianz im Leseverstehen (R) nicht durch D und C aufgeklärt werden konnte, deutet darauf hin, dass das Modell nicht vollständig ist und ggf. durch weitere Komponenten ergänzt werden muss. Studie 3 untersucht die Verarbeitung positiv-kausaler und negativ-kausaler Kohärenzrelationen bei deutschen Erst- bis Viertklässler(inne)n und Erwachsenen im Lese- und Hörverstehen. In Übereinstimmung mit dem Cumulative Cognitive Complexity-Ansatz (Evers-Vermeul & Sanders, 2009; Spooren & Sanders, 2008) zeigt Studie 3, dass die Verarbeitung negativ-kausaler Kohärenzrelationen und Konnektoren kognitiv aufwändiger ist als die Verarbeitung positiv-kausaler Relationen. Darüber hinaus entwickelt sich das Verstehen beider Kohärenzrelationen noch über die Grundschulzeit hinweg und ist für negativ-kausale Relationen am Ende der vierten Klasse noch nicht abgeschlossen. Studie 4 zeigt und diskutiert die Nützlichkeit prozess-orientierter Lesetests wie ProDi- L (Richter et al., in press), die individuelle Unterschiede in den kognitiven Teilfertigkeiten des Leseverstehens selektiv erfassen. Hierzu wird exemplarisch die Konstruktvalidität des ProDi-L-Subtests ‚Syntaktische Integration’ nachgewiesen. Mittels explanatorischer Item- Repsonse-Modelle wird gezeigt, dass der Test Fertigkeiten syntaktischer Integration separat erfasst und Kinder mit defizitären syntaktischen Fertigkeiten identifizieren kann. Die berichteten Befunde tragen zu einem umfassenden Verständnis der kognitiven Teilfertigkeiten des Leseverstehens bei, das für eine optimale Gestaltung des Leseunterrichts, für das Erstellen von Lernmaterialien, Leseinstruktionen und Lehrbüchern unerlässlich ist. Darüber hinaus stellt es die Grundlage für eine sinnvolle Diagnose individueller Leseschwierigkeiten und für die Konzeption adaptiver und zielgerichteter Interventionsprogramme zur Förderung des Leseverstehens bei schwachen Leser(inne)n dar.

RESILIENCE OF NETWORKED INFRASTRUCTURE WITH EVOLVING COMPONENT CONDITIONS: A PAVEMENT NETWORK APPLICATION

This thesis deals with quantifying the resilience of a network of pavements. Calculations were carried out by modeling network performance under a set of possible damage-meteorological scenarios with known probability of occurrence. Resilience evaluation was performed a priori while accounting for optimal preparedness decisions and additional response actions that can be taken under each of the scenarios. Unlike the common assumption that the pre-event condition of all system components is uniform, fixed, and pristine, component condition evolution was incorporated herein. For this purpose, the health of the individual system components immediately prior to hazard event impact, under all considered scenarios, was associated with a serviceability rating. This rating was projected to reflect both natural deterioration and any intermittent improvements due to maintenance. The scheme was demonstrated for a hypothetical case study involving Laguardia Airport. Results show that resilience can be impacted by the condition of the infrastructure elements, their natural deterioration processes, and prevailing maintenance plans. The findings imply that, in general, upper bound values are reported in ordinary resilience work, and that including evolving component conditions is of value.

TEST-ANALYSIS MODEL CORRELATION AND CALIBRATION OF A MULTI-COMPONENT PLASTIC PART

The goal of this project is to learn the necessary steps to create a finite element model, which can accurately predict the dynamic response of a Kohler Engines Heavy Duty Air Cleaner (HDAC). This air cleaner is composed of three glass reinforced plastic components and two air filters. Several uncertainties arose in the finite element (FE) model due to the HDAC’s component material properties and assembly conditions. To help understand and mitigate these uncertainties, analytical and experimental modal models were created concurrently to perform a model correlation and calibration. Over the course of the project simple and practical methods were found for future FE model creation. Similarly, an experimental method for the optimal acquisition of experimental modal data was arrived upon. After the model correlation and calibration was performed a validation experiment was used to confirm the FE models predictive capabilities.

Bayesian Estimation of Small Proportions Using Binomial Group Test

Group testing has long been considered as a safe and sensible relative to one-at-a-time testing in applications where the prevalence rate p is small. In this thesis, we applied Bayes approach to estimate p using Beta-type prior distribution. First, we showed two Bayes estimators of p from prior on p derived from two different loss functions. Second, we presented two more Bayes estimators of p from prior on π according to two loss functions. We also displayed credible and HPD interval for p. In addition, we did intensive numerical studies. All results showed that the Bayes estimator was preferred over the usual maximum likelihood estimator (MLE) for small p. We also presented the optimal β for different p, m, and k.

The Herpesvirus Nuclear Egress Complex Component, UL31, is Recruited to Sites of DNA Damage Through Poly(ADP-RIBOSE) Binding

Thesis (Master, Biomedical & Molecular Sciences) -- Queen's University, 2016-08-23 15:03:30.807

A principal component approach to space-based gravitational wave astronomy

The current approach to data analysis for the Laser Interferometry Space Antenna (LISA) depends on the time delay interferometry observables (TDI) which have to be generated before any weak signal detection can be performed. These are linear combinations of the raw data with appropriate time shifts that lead to the cancellation of the laser frequency noises. This is possible because of the multiple occurrences of the same noises in the different raw data. Originally, these observables were manually generated starting with LISA as a simple stationary array and then adjusted to incorporate the antenna's motions. However, none of the observables survived the flexing of the arms in that they did not lead to cancellation with the same structure. The principal component approach is another way of handling these noises that was presented by Romano and Woan which simplified the data analysis by removing the need to create them before the analysis. This method also depends on the multiple occurrences of the same noises but, instead of using them for cancellation, it takes advantage of the correlations that they produce between the different readings. These correlations can be expressed in a noise (data) covariance matrix which occurs in the Bayesian likelihood function when the noises are assumed be Gaussian. Romano and Woan showed that performing an eigendecomposition of this matrix produced two distinct sets of eigenvalues that can be distinguished by the absence of laser frequency noise from one set. The transformation of the raw data using the corresponding eigenvectors also produced data that was free from the laser frequency noises. This result led to the idea that the principal components may actually be time delay interferometry observables since they produced the same outcome, that is, data that are free from laser frequency noise. The aims here were (i) to investigate the connection between the principal components and these observables, (ii) to prove that the data analysis using them is equivalent to that using the traditional observables and (ii) to determine how this method adapts to real LISA especially the flexing of the antenna. For testing the connection between the principal components and the TDI observables a 10x 10 covariance matrix containing integer values was used in order to obtain an algebraic solution for the eigendecomposition. The matrix was generated using fixed unequal arm lengths and stationary noises with equal variances for each noise type. Results confirm that all four Sagnac observables can be generated from the eigenvectors of the principal components. The observables obtained from this method however, are tied to the length of the data and are not general expressions like the traditional observables, for example, the Sagnac observables for two different time stamps were generated from different sets of eigenvectors. It was also possible to generate the frequency domain optimal AET observables from the principal components obtained from the power spectral density matrix. These results indicate that this method is another way of producing the observables therefore analysis using principal components should give the same results as that using the traditional observables. This was proven by fact that the same relative likelihoods (within 0.3%) were obtained from the Bayesian estimates of the signal amplitude of a simple sinusoidal gravitational wave using the principal components and the optimal AET observables. This method fails if the eigenvalues that are free from laser frequency noises are not generated. These are obtained from the covariance matrix and the properties of LISA that are required for its computation are the phase-locking, arm lengths and noise variances. Preliminary results of the effects of these properties on the principal components indicate that only the absence of phase-locking prevented their production. The flexing of the antenna results in time varying arm lengths which will appear in the covariance matrix and, from our toy model investigations, this did not prevent the occurrence of the principal components. The difficulty with flexing, and also non-stationary noises, is that the Toeplitz structure of the matrix will be destroyed which will affect any computation methods that take advantage of this structure. In terms of separating the two sets of data for the analysis, this was not necessary because the laser frequency noises are very large compared to the photodetector noises which resulted in a significant reduction in the data containing them after the matrix inversion. In the frequency domain the power spectral density matrices were block diagonals which simplified the computation of the eigenvalues by allowing them to be done separately for each block. The results in general showed a lack of principal components in the absence of phase-locking except for the zero bin. The major difference with the power spectral density matrix is that the time varying arm lengths and non-stationarity do not show up because of the summation in the Fourier transform.

Modeling, Component Selection and Optimization of Servo-controlled Automatic Machinery

A servo-controlled automatic machine can perform tasks that involve synchronized actuation of a significant number of servo-axes, namely one degree-of-freedom (DoF) electromechanical actuators. Each servo-axis comprises a servo-motor, a mechanical transmission and an end-effector, and is responsible for generating the desired motion profile and providing the power required to achieve the overall task. The design of a such a machine must involve a detailed study from a mechatronic viewpoint, due to its electric and mechanical nature. The first objective of this thesis is the development of an overarching electromechanical model for a servo-axis. Every loss source is taken into account, be it mechanical or electrical. The mechanical transmission is modeled by means of a sequence of lumped-parameter blocks. The electric model of the motor and the inverter takes into account winding losses, iron losses and controller switching losses. No experimental characterizations are needed to implement the electric model, since the parameters are inferred from the data available in commercial catalogs. With the global model at disposal, a second objective of this work is to perform the optimization analysis, in particular, the selection of the motor-reducer unit. The optimal transmission ratios that minimize several objective functions are found. An optimization process is carried out and repeated for each candidate motor. Then, we present a novel method where the discrete set of available motor is extended to a continuous domain, by fitting manufacturer data. The problem becomes a two-dimensional nonlinear optimization subject to nonlinear constraints, and the solution gives the optimal choice for the motor-reducer system. The presented electromechanical model, along with the implementation of optimization algorithms, forms a complete and powerful simulation tool for servo-controlled automatic machines. The tool allows for determining a wide range of electric and mechanical parameters and the behavior of the system in different operating conditions.

Doehlert Design-desirability Function Multi-criteria Optimal Separation Of 17 Phenolic Compounds From Extra-virgin Olive Oil By Capillary Zone Electrophoresis.

In Brazil, the consumption of extra-virgin olive oil (EVOO) is increasing annually, but there are no experimental studies concerning the phenolic compound contents of commercial EVOO. The aim of this work was to optimise the separation of 17 phenolic compounds already detected in EVOO. A Doehlert matrix experimental design was used, evaluating the effects of pH and electrolyte concentration. Resolution, runtime and migration time relative standard deviation values were evaluated. Derringer's desirability function was used to simultaneously optimise all 37 responses. The 17 peaks were separated in 19min using a fused-silica capillary (50μm internal diameter, 72cm of effective length) with an extended light path and 101.3mmolL(-1) of boric acid electrolyte (pH 9.15, 30kV). The method was validated and applied to 15 EVOO samples found in Brazilian supermarkets.

Two-component System Vicrk Regulates Functions Associated With Establishment Of Streptococcus Sanguinis In Biofilms.

Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK in S. sanguinis (VicRKSs), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. A vicK knockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H2O2, a metabolic product associated with DNA release, and with inhibitory capacity of S. sanguinis competitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094, cwdP, and gbpB) and spxB, which encodes pyruvate oxidase for H2O2 production. Genes previously associated with spxB expression (spxR, ccpA, ackA, and tpK) were not transcriptionally affected in SKvic. RT-qPCR analyses of S. sanguinis biofilm cells further showed upregulation of VicRK targets (spxB, gbpB, and SSA_0094) and other genes for biofilm formation (gtfP and comE) compared to expression in planktonic cells. This study provides evidence that VicRKSs regulates functions crucial for S. sanguinis establishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.

Control Of The Properties Of Porous Chitosan-alginate Membranes Through The Addition Of Different Proportions Of Pluronic F68.

This work addresses the development and characterization of porous chitosan-alginate based polyelectrolyte complexes, obtained by using two different proportions of the biocompatible surfactant Pluronic F68. These biomaterials are proposed for applications as biodegradable and biocompatible wound dressing and/or scaffolds. The results indicate that thickness, roughness, porosity and liquid uptake of the membranes increase with the amount of surfactant used, while their mechanical properties and stability in aqueous media decrease. Other important properties such as color and surface hydrophilicity (water contact angle) are not significantly altered or did not present a clear tendency of variation with the increase of the amount of surfactant added to the polyelectrolyte complexes, such as real density, average pore diameter, total pore volume and surface area. The prepared biomaterials were not cytotoxic to L929 cells. In conclusion, it is possible to tune the physicochemical properties of chitosan-alginate polyelectrolyte complexes, through the variation of the proportion of surfactant (Pluronic F68) added to the mixture, so as to enable the desired application of these biomaterials.

Sci1 Is A Component Of The Auxin-dependent Control Of Cell Proliferation In Arabidopsis Upper Pistil.

To characterize the recently described SCI1 (stigma/style cell cycle inhibitor 1) gene relationship with the auxin pathway, we have taken the advantage of the Arabidopsis model system and its available tools. At first, we have analyzed the At1g79200 T-DNA insertion mutants and constructed various transgenic plants. The loss- and gain-of-function plants displayed cell number alterations in upper pistils that were controlled by the amino-terminal domain of the protein. These data also confirmed that this locus holds the functional homolog (AtSCI1) of the Nicotiana tabacum SCI1 gene. Then, we have provided some evidences the auxin synthesis/signaling pathways are required for downstream proper AtSCI1 control of cell number: (a) its expression is downregulated in yuc2yuc6 and npy1 auxin-deficient mutants, (b) triple (yuc2yuc6sci1) and double (npy1sci1) mutants mimicked the auxin-deficient phenotypes, with no synergistic interactions, and (c) the increased upper pistil phenotype in these last mutants, which is a consequence of an increased cell number, was able to be complemented by AtSCI1 overexpression. Taken together, our data strongly suggests SCI1 as a component of the auxin signaling transduction pathway to control cell proliferation/differentiation in stigma/style, representing a molecular effector of this hormone on pistil development.