Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.

ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity.

The cleavage specificity of the Pvu II and BamHI restriction endonucleases is found to be dramatically reduced at elevated osmotic pressure. Relaxation in specificity of these otherwise highly accurate and specific enzymes, previously termed "star activity," is uniquely correlated with osmotic pressure between 0 and 100 atmospheres. No other colligative solvent property exhibits a uniform correlation with star activity for all of the compounds tested. Application of hydrostatic pressure counteracts the effects of osmotic pressure and restores the natural selectivity of the enzymes for their canonical recognition sequences. These results indicate that water solvation plays an important role in the site-specific recognition of DNA by many restriction enzymes. Osmotic pressure did not induce an analogous effect on the specificity of the EcoRV endonuclease, implying that selective hydration effects do not participate in DNA recognition in this system. Hydrostatic pressure was found to have little effect on the star activity induced by changes in ionic strength, pH, or divalent cation, suggesting that distinct mechanisms may exist for these observed alterations in specificity. Recent evidence has indicated that BamHI and EcoRI share similar structural motifs, while Pvu II and EcoRV belong to a different structural family. Evidently, the use of hydration water to assist in site-specific recognition is a motif neither limited to nor defined by structural families.

The Impact of Shyness on Loneliness, Social Anxiety, and School Liking in Late Childhood

Author: Charity M. Walker Title: THE IMPACT OF SHYNESS ON LONELINESS, SOCIAL ANXIETY, AND SCHOOL LIKING IN LATE CHILDHOOD Advisor: Maria T. Riva, Ph.D. Degree Date: August 2011 ABSTRACT Shyness is associated with several emotional, social, and academic problems. While there are multiple difficulties that often accompany shyness, there appear to be some factors that can moderate negative effects of shyness. Research has demonstrated that certain parenting factors affect the adjustment of shy children in early childhood, but there is minimal research illuminating the effect of parenting factors in older age groups. The first purpose of this study was to examine relationships between shyness and loneliness, social anxiety, and school liking. The second purpose was to investigate whether the quality of the relationship between a parent and a 10- to 15-year-olds child influences the amount of loneliness or social anxiety a shy child experiences or how the child feels about school. Parent-child dyads served as participants and were recruited from public and private middle schools and church youth groups in Colorado and Indiana. Child participants completed several self-report surveys regarding their relationship with a parent, shyness, loneliness, social anxiety, and their attitude toward school. Parents completed a survey about their relationship with their child and responded to questions related to their perceptions of their child's shyness. Data was analyzed with a series of correlation and regression analyses. Greater degrees of self-reported shyness were found to be associated with higher levels of loneliness and social anxiety and less positive feelings about school. Due to a problem with multicollinearity during data analysis, this study was not able to explore the effect of the parent-child relationship quality on the associations between shyness and adjustment factors. Overall, these findings imply that shyness remains an important issue as children approach adolescence. Further research is needed to continue learning about the potential importance of parent-child interactions in reducing maladjustment for shy children during late childhood.

A Qualitative Study on Successful African American Marriages on How They Create and Repair Attachment Injuries Using Emotional Skillfulness.

The purpose of this phenomenological study was to describe the meanings that Ossie Davis and Ruby Dee Davis attribute to how they have adapted to their marriage and overcame conflict over the years. A purposive sample of this well-known African American married couple, who self-identified as being in a long-term, successful marriage, was used. The subjects of study were married for 56 years. An analysis using Colaizzi's (1978) method revealed 2 themes, with 13 and 2 subthemes respectively. The themes that emerged from the analysis of the formulated meanings were: (1) secrets to a successful marriage; and (2) sources of conflict in marriage. Secrets to a successful marriage included 13 subthemes: (a) egalitarian roles; (b) commitment; (c) forgiveness; (d) communication; (e) love; (f) honesty; (g) understanding the struggles of Black males and females; (h) friendship; (i) religion/ support from God; (j) compromise; (k) beliefs that marriage is a process; (l) emotional availability of spouse; and (m) feelings of security. In addition, the theme sources of conflict in marriage had two subthemes: (a) different decision making styles; and (b) experiences of abandonment. These findings provided insight from this couple's perspective on the secrets to a successful marriage and the ways in which they managed to make their marriage work, in light of the unique challenges that face African American marriages.

Performance Septic Systems: Does an Advanced Septic System Provide Adequate Treatment on Black Hammock Island and is there Adequate Regulatory Policy to Ensure Integrity?

This project determined if the performance based septic system, proposed for a development on Black Hammock Island in Jacksonville, Florida, is the best choice to prevent environmental contamination from septic tanks and if adequate regulatory policy exists. Research shows a central wastewater treatment plant provides the most efficient pollutant removal but can be cost prohibitive. This project found performance based systems surpasses conventional septic system performance and are more suited and economical to remote areas without sewer service. This project finds current regulations to be inadequate and supports ordinance changes proposed by the Mayor of Jacksonville to enhance the current policy and provide more adequate and meaningful regulation.

Effect of carbon fibres on the mechanical properties and corrosion levels of reinforced portland cement mortars

The changes in mechanical properties of portland cement mortars due to the addition of carbon fibres (CF) to the mix have been studied. Compression and flexural strengths have been determined in relation to the amount of fibres added to the mix, water/binder ratio, curing time and porosity. Additionally, the corrosion level of reinforcing steel bars embedded in portland cement mortars containing CF and silica fume (SF) have also been investigated and reinforcing steel corrosion rates have been determined. As a consequence of the large concentration of oxygen groups in CF surface, a good interaction between the CF and the water of the mortar paste is to be expected. A CF content of 0.5% of cement weight implies an optimum increase in flexural strength and an increase in embedded steel corrosion.

The imprint of the crustal magnetic field on the thermal spectra and pulse profiles of isolated neutron stars

Isolated neutron stars (NSs) show a bewildering variety of astrophysical manifestations, presumably shaped by the magnetic field strength and topology at birth. Here, using state-of-the-art calculations of the coupled magnetic and thermal evolution of NSs, we compute the thermal spectra and pulse profiles expected for a variety of initial magnetic field configurations. In particular, we contrast models with purely poloidal magnetic fields to models dominated by a strong internal toroidal component. We find that, while the former displays double-peaked profiles and very low pulsed fractions, in the latter, the anisotropy in the surface temperature produced by the toroidal field often results in a single pulse profile, with pulsed fractions that can exceed the 50–60 per cent level even for perfectly isotropic local emission. We further use our theoretical results to generate simulated ‘observed’ spectra, and show that blackbody (BB) fits result in inferred radii that can be significantly smaller than the actual NS radius, even as low as ∼1–2 km for old NSs with strong internal toroidal fields and a high absorption column density along their line of sight. We compute the size of the inferred BB radius for a few representative magnetic field configurations, NS ages and magnitudes of the column density. Our theoretical results are of direct relevance to the interpretation of X-ray observations of isolated NSs, as well as to the constraints on the equation of state of dense matter through radius measurements.

Development of novel nano-biocomposite antioxidant films based on poly (lactic acid) and thymol for active packaging

Novel nano-biocomposite films based on poly (lactic acid) (PLA) were prepared by incorporating thymol, as the active additive, and modified montmorillonite (D43B) at two different concentrations. A complete thermal, structural, mechanical and functional characterization of all nano-biocomposites was carried out. Thermal stability was not significantly affected by the addition of thymol, but the incorporation of D43B improved mechanical properties and reduced the oxygen transmission rate by the formation of intercalated structures, as suggested by wide angle X-ray scattering patterns and transmission electron microscopy images. The addition of thymol decreased the PLA glass transition temperature, as the result of the polymer plasticization, and led to modification of the elastic modulus and elongation at break. Finally, the amount of thymol remaining in these formulations was determined by liquid chromatography (HPLC-UV) and the antioxidant activity by the DPPH spectroscopic method, suggesting that the formulated nano-biocomposites could be considered a promising antioxidant active packaging material.

The influence of surfactant loading level in a montmorillonite on the thermal, mechanical and rheological properties of EVA nanocomposites

In this work, montmorillonite (Mt) has been organically modified with ethyl hexadecyl dimethyl ammonium (EHDDMA) in 20, 50, 80 and 100% of the nominal exchange capacity (CEC) of the Mt. A full characterization of the organo-montmorillonite (OMt) obtained has been made, including thermal analysis, X-Ray Diffraction, elemental analysis CHN and nitrogen adsorption. According to the results, 12% in mass of the surfactant added is strongly retained by the Mt. When the mass percentage of EHDDMA exchanged in the OMt is increased up to this level, the interactions OMt–EHDDMA are steeply reduced depending on the EHDDMA content. Clay polymer nanocomposites (CPN) were prepared by melt mixing of EVA and different loads of OMt. The CPN were compress molded to obtain 1 mm thick sheets, which have been characterized according to their mechanical, thermal and rheological behaviors. The major changes in the structure of the OMt are obtained for low contents of EHDDMA. Nevertheless, the CPN containing OMt exchanged at 20 and 50% of the CEC show relatively low effect of the EHDDMA while the mechanical response and rheological behavior of CPN with OMt modified at 80 and 100% of the CEC are much more pronounced.

Current status of research on school refusal

School refusal behavior refers to the avoidance of a child attending school and/or persistent difficulty staying in the classroom throughout the school day. Based on a review of the scientific literature, the aim of this study is to describe the current state of research on school refusal, differentiating between the findings and progress made in Spain from those achieved in the international field. For this purpose, the significance of this phenomenon, in addition to associated risk factors and variables, will be reviewed in the child and youth population. In turn, the commonly used assessment methods and most recommended treatment proposals, mainly based on cognitive behavioral therapy, are discussed. The results reveal several gaps and subjects for debate in some areas of knowledge about school refusal behavior, with differences being found between Spanish and international studies. In conclusion, future studies and challenges in this field are required.

Experimental research on the residual mechanical properties of an ordinary concretes after fire

This paper summarizes the results of an experimental research to assess the residual mechanical properties of an ordinary concrete after fire. It was studied the influence of the cooling process, the maximum temperature that the concrete was subjected to and the loading level on the residual mechanical properties of calcareous and granite aggregate concretes. The properties studied were the residual compressive, tensile, splitting and flexural strengths and modulus of elasticity. Four levels of temperature; 20, 300, 500 and 700ºC; two loading levels (0.3f and 0.7fcd) and two cooling processes (cooling in the air and by water jet) were tested. The high temperatures attained and the sudden cooling down process on the concrete showed a negative effect on its residual mechanical properties. This effect was more notorious on the residual compression strength than in the other mechanical properties