Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

A study of organizational citizenship behaviors in a retail setting

This study investigates the potential antecedents of organizational citizenship behaviors (OCBs) in a retail setting. Much remains unknown about the factors affecting OCBs in retail settings. Several characteristics of retail jobs, as compared with other organizational behavior contexts, suggest the need to examine antecedents of OCBs. Job attitudes (job satisfaction and organizational commitment) are proposed as direct predictors of OCBs. Leadership support, professional development, and empowerment are posited as indirect predictors of OCBs and direct predictors of job attitudes. The possible moderating impacts of employee demographics and job types on the modeled relationships are also examined. The research hypotheses are tested using data collected from 211 frontline employees who work in a retail setting. The employees have customer-contact roles in the upscale food and grocery retailer that participated in the study. The pattern of results is more complex than hypothesized. Job attitudes are related to OCBs but the mediating role of job attitudes is not supported. The relationships between leadership support, professional development, and empowerment, and OCBs and job attitudes differ systematically. Evidence of how employee demographics can alter the modeled relationships is also presented. The findings have significant implications for the theory and practice of managing frontline employees. Limitations of the study are discussed and a program of further research is sketched. © 2003 Elsevier Inc. All rights reserved.

Generation of nitric oxide and its protective and toxic actions in the gastrointestinal tract

Nitric oxide is a free-radical gas which can exert both protective and damaging effects. The objectives of the thesis were: (i) to investigate arginine metabolism in isolated rat gastric mucosal cells, (ii) to investigate the role of NO in the induction of ornithine decarboxylase in the rat gastric mucosa damaged by hypertonic saline in vivo, (iii) to expose primary cultures of guinea-pig gastric mucosal cells to oxidative challenge and an NO donor, and to investigate the response in terms of heat shock protein 72 (HSP 72) induction, and (iv) to investigate the induction of iNOS and the role of potential modulators of activity in gastric cell lines. Isolated rat gastric mucosal cells converted exogenous arginine to ornithine and citrulline. This metabolism of arginine was not affected by a range of NO synthase inhibitors, but was reduced by the arginase inhibitors NG-hydroxy-L-arginine and L-ornithine. Thus, the predominant pathway of arginine metabolism involves arginase and ornithine transcarbamoylase, not NO synthase. Pretreatment of rats with NG-nitro-L-arginine promoted activation of ornithine decarboxylase after intragastric hypertonic saline, but did not increase acid phosphatase release (damage). NO may therefore restrict activation of ornithine decarboxylase in response to damage. Exposure of primary cultures of guinea-pig gastric mucosal cells to S-nitroso-N-acetyl-penicillamine (SNAP) caused a concentration dependent induction of HSP 72, which was inhibited by an NO scavenger and blockade of transcription. The effect of SNAP was enhanced by decreasing the intracellular reduced thiol content with diethyl maleate, which itself also induced HSP 72 formation. Substantial amounts of NO may induce defensive responses in cells. Induction of iNOS was not detected in HGT-1 or AGS cells exposed to cytokines. Conclusions An arginase pathway may restrict availability of arginine for NO synthase in gastric mucosa or may be present to supply ornithine for polyamine synthesis. NO may modulate the response to damage of the stomach epithelium in vivo. Exogenous NO may induce a defensive response in gastric mucosal cells.

Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox

Hypochlorous acid (HOCl) concentration-dependently decreased ATPase activity and SH groups of pure Ca-ATPase from sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle with IC(50) of 150 micromol/l and 6.6 micromol/l, respectively. This indicates that SH groups were not critical for impairment of Ca-ATPase activity. Pure Ca-ATPase activity was analysed individually with respect to both substrates, Ca(2+) and ATP. Concerning dependence of ATPase activity on HOCl (150 micromol/l) as a function of free Ca(2+) and ATP, V(max) of both dependences decreased significantly, while the affinities to individual substrates were not influenced, with the exception of the regulatory binding site of ATP. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) decreased, indicating binding of HOCl to nucleotide binding site of SERCA. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Fragmentation may be associated with protein carbonyl formation. The density of protein carbonyl bands at 75 and 110 kDa increased concentration- and time-dependently. Trolox (250 micromol/l) recovered the Ca-ATPase activity decrease induced by HOCl, probably by changing conformational properties of the Ca-ATPase protein. Trolox inhibited FITC binding to SERCA.

Discriminating behaviors through value added premium online grocery offering

Despite many interest in e-grocery, little has changed, over the years, in the offering that is often geared only towards low value staple products. Yet, from an e-supermarket perspective, the number of sourcing stores is increasing regularly providing an illusion of service improvement. This situation, we argue is leading e-grocery providers to forego profits as consumers need to look both at the competition online and offline to satisfy their overall regular grocery needs. Expansion of e-grocery operations could be better achieved, we argue, by serving diverse and premium priced products (e.g. organic, limited production, regional items; special occasions items and products related to health e.g. allergies, diabetes) and utilizing more efficiently modern logistic techniques. A framework is offered presenting a model including the delivery of premium products from various suppliers and providing an integrated service solution to e-grocery customers that complete traditional supermarket ranges, creating potential high value added products niches. In this context, the objective was to understand the consumer discrimination factors (ie: range of product, delivery timing, location, service quality) leading to intentions towards purchasing more items from e-grocery retailers. Data are derived from a survey of 356 respondents in Turkey’s three biggest metropolitan areas. The relationship between consumer attitudes and demographic characteristics are also analyzed. Factor and SEM analyses are used to discriminate within the sample (n=356, no of items=150). Results, future research and policy implications are discussed.

Induction of placental heme oxygenase-1 is protective against TNFα-induced cytotoxicity and promotes vessel relaxation

Background: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. Materials and Methods: We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNF) induced placental damage and on feto-placental circulation was studied. Results: We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNF-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Conclusions: Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

The mediating role of discrete emotions in the relationship between injustice and counterproductive work behaviors:a study in Pakistan

Purpose: Our study explores the mediating role of discrete emotions in the relationships between employee perceptions of distributive and procedural injustice, regarding an annual salary raise, and counterproductive work behaviors (CWBs). Design/Methodology/Approach: Survey data were provided by 508 individuals from telecom and IT companies in Pakistan. Confirmatory factor analysis, structural equation modeling, and bootstrapping were used to test our hypothesized model. Findings: We found a good fit between the data and our tested model. As predicted, anger (and not sadness) was positively related to aggressive CWBs (abuse against others and production deviance) and fully mediated the relationship between perceived distributive injustice and these CWBs. Against predictions, however, neither sadness nor anger was significantly related to employee withdrawal. Implications: Our findings provide organizations with an insight into the emotional consequences of unfair HR policies, and the potential implications for CWBs. Such knowledge may help employers to develop training and counseling interventions that support the effective management of emotions at work. Our findings are particularly salient for national and multinational organizations in Pakistan. Originality/Value: This is one of the first studies to provide empirical support for the relationships between in/justice, discrete emotions and CWBs in a non-Western (Pakistani) context. Our study also provides new evidence for the differential effects of outward/inward emotions on aggressive/passive CWBs. © 2012 Springer Science+Business Media, LLC.

Retrospective reports of child feeding practices, current eating behaviors, and BMI in college students

Research concerning child feeding practices has focused on children and adolescents, and little is known about how feeding practices used in childhood relate to eating behaviors and weight status in early adulthood. We assessed college students' and their parents' retrospective reports of child feeding practices used when the students were in middle childhood. We also assessed the college students' current reports of their eating behaviors using the Dutch Eating Behavior Questionnaire (DEBQ) and the Intuitive Eating Scale (IES), and measured their current BMI. Results showed that college students' and their parents' reports about previous parental use of child feeding practices were not correlated. Parent reports of their own use of child feeding practices were more related to students' eating behaviors and BMI than were students' recollections about feeding practices used by their parents. An analysis of gender effects showed that there were positive correlations between parental child feeding practices, BMI, and emotional eating for female students. These relationships did not exist for male students. The results suggest that child feeding practices recollected by parents are linked to the development of emotional eating and weight status of women in early adulthood.

Work-life balance benefits:employee attitudes and behaviors through the lens of social exchange theory

The question of what to provide employees in order that they reciprocate with desirable behaviors in the work place has resulted in a great amount of work in the area of social exchange. Although offering fair compensation, including salary or wages and employee benefits, has been extensively studied, the effects of offering specific types of benefits, such as work-life balance benefits, and the intangible rewards that such an offering inadvertently offers, has only been minimally explored. Utilizing past literature, this current research examined the offering of work-life balance benefits, the value employees place on those benefits, the communication of the benefits by the organization to employees, and their effect on employee attitudes and behaviors. The goal was to identify the effect on desirable outcomes when work-life balance benefits are offered to determine the usefulness to the organization of offering such benefits. To test these effects, a study of an organization known to offer a strong work-life balance benefits package was undertaken. This was accomplished through the distribution of questionnaires to identify the possible relationships involving 408 employee respondents and their 79 supervisors. This was followed with interviews of 12 individuals to ascertain the true reasons for links observed through analysis. Analysis of the data was accomplished through correlation analysis, multilevel analysis and regression analysis generated by SPSS. The results of the quantitative analysis showed support for a relationship between the offering of work-life balance benefits and perceived organizational support, perceived distributive justice, job satisfaction and OCBO. The analysis also showed a lack of support for a relationship between the offering of work-life balance benefits and organizational commitment, OCBI and IRB. The interviews offered possible reasons for the lack of support regarding the relationship between the offering of work-life balance benefits and organizational commitment as well as organizational citizenship behaviors (OCBI and IRB). The implications of these findings on future research, theory and practice in the offering of work-life balance benefits are discussed.

Do parental feeding practices moderate the relationships between impulsivity and eating in children?

This study examines the relationships between children's impulsivity, their eating behaviours, and their perceptions of their parent's feeding practices. 153 10-13. year old children completed questionnaires assessing their eating behaviours, their impulsiveness and their perception of their parent's feeding practices. Children's reports of dysfunctional eating behaviours were significantly correlated with their perceptions of their parents feeding practices and with their levels of impulsivity. Children's reports of parental monitoring of their food intake significantly moderated the influence of child impulsiveness upon emotional eating. Children's perceptions of parental monitoring of their food intake may potentially have a protective effect at preventing more impulsive children from eating in response to emotional feelings. © 2011 Elsevier Ltd.