Optimization Of Promoter Sequences In Order To Mimic Physiological Pde6b Expression

Purpose: Many retinal degenerations result from defective retina-specific gene expressions. Thus, it is important to understand how the expression of a photoreceptor-specific gene is regulated in vivo in order to achieve successful gene therapy. The present study aims to design an AAV2/8 vector that can regulate the transcript level in a physiological manner to replace missing PDE6b in Rd1 and Rd10 mice. In previous studies (Ogieta, et al., 2000), the short 5' flanking sequence of the human PDE6b gene (350 bp) was shown to be photoreceptor-specific in transgenic mice. However, the efficiency and specificity of the 5' flanking region of the human PDE6b was not investigated in the context of gene therapy during retinal degeneration. In this study, two different sequences of the 5' flanking region of the human PDE6b gene were studied as promoter elements and their expression will be tested in wild type and diseased retinas (Rd 10 mice).Methods: Two 5' flanking fragments of the human PDE6b gene: (-93 to +53 (150 bp) and -297 to +53 (350 bp)) were cloned in different plasmids in order to check their expression in vitro and in vivo by constructing an AAV2/8 vector. These elements drove the activity of either luciferase (pGL3 plasmids) or EGFP. jetPEI transfection in Y 79 cells was used to evaluate gene expression through luciferase activity. Constructs encoding EGFP under the control of the two promoters were performed in AAV2.1-93 (or 297)-EGFP plasmids to produce AAV2/8 vectors.Results: When pGL3-93 (150 bp) or pGL3-297 (350 bp) were transfected in the Y-79 cells, the smaller fragment (150 bp) showed higher gene expression compared to the 350 bp element and to the SV40 control, as previously reported. The 350 bp drove similar levels of expression when compared to the SV40 promoter. In view of these results, the fragments (150 bp or 350 bp) were integrated into the AAV2.1-EGFP plasmid to produce AAV2/8 vector, and we are currently evaluating the efficiency and specificity of the produced constructs in vivo in normal and diseased retinas.Conclusions: Comparisons of these vectors with vectors bearing ubiquitous promoters should reveal which construct is the most suitable to drive efficient and specific gene expression in diseased retinas in order to restore a normal function on the long term.

Cuando Frankenstein encontró a Dorian Gray: dandysmo, post-identidad y sujetos virtuales

The appearance and popularization of the internet has created new forms of writing, which compel us to think anew about identity and subjectivity. Webjournals or blogs are specially interesting because they are a massive phenomenon that use autobiographical writing in a peculiar way. These forms of writing stress a particular paradox of the genre: the coexistence between a purpose of private, confessional and spontaneous writing and a public image, carefully built, as a result of its writing. The technology is new, but, in fact, the paradox is old. This paper tries to explore this old paradox, our eternal condition of cyborgs, our use of technologies in order to construct a public, unique and recognizably identity. In oder to do so, I will try to show the virtual condition of any written individual ¿this issue has already been dealt with by autobiographical studies¿, focusing on blogs, and especially on concrete example (Lord Whimsy¿s Journal). I will pay attention to gender as a technology that constructs identity and, at the same time, is deconstructed by the autobiographical narratives analyzed. In short, I attempt to show that virtual and autobiographical discourse do not bring forth a new kind of subject but the permanence of an old phenomenon "clearly developed by dandyism, for instance¿: the use of tehnologies to re-invent, re-formulate and re-construct us as multiple, hybrid and mixed subjects."

Analytical Measures of Learners' Written Interlanguage

This paper aims at reconsidering some analytical measures to best encapsulate the interlanguage, in writing, of young beginner learners of English as a foreign language in the light of previous and work-in-progress research conducted within the BAF project, and in particular, whether clause and sentence length should be best viewed as a fluency or syntactic complexity measusre or as part of a different construct. In the light of a factor analysis (Navés, forthcoming) and multivariate and correlation studies (Navés et al. 2003, Navés, 2006, Torres et al. 2006) it becomes clear that the relationship between different analytical measures is also dependent on learner¿s cognitive maturity (age) and proficiency (amount of instruction). Finally, clause and sentence length should not be viewed as either a fluency or sytactic complexity measure but as part of a different construct. It is concluded that further research using regression analysis and cluster analysis is neeed in order to identify and validate the constructs of the writing components and their measurements.

Small RNA-dependent expression of secondary metabolism is controlled by Krebs cycle function in Pseudomonas fluorescens.

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.

Prosody and pragmatics in parenthetical insertions in catalan

This paper analyses the role of prosody in parenthetical insertions, a type of structure that is extremely common in both speech and writing. The materials under study come from a corpus of spontaneous speech acts in Central Catalan (with varying degrees of spontaneity) from which a corpus of oral parenthetical insertions has been compiled. The prototypical prosodic features of a parenthetical insertion in Catalan are: prosodic autonomy, limited extension, production in between pauses or final pause, tendency towards acceleration, fall in intensity, lower pitch range and, finally, falling or rising melodic pattern. While the final fall is the most frequent pattern in spontaneous conversations with a high degree of confidence between interlocutors, a final rising structure is found in interviews in which the degree of confidence between participants is smaller, their roles are unequal, and the interviewed constructs a narrative discourse. We thus suggest that the pitch contour of parenthetical insertions is related to formality and discourse typology (in this case, narrative vs. dialogue). Bearing in mind the discursive functions performed by these insertions, we propose a typology which classifies them with regards to two main functions: completion of information, and modalisation.

Salicylic Acid Biosynthetic Genes Expressed in Pseudomonas fluorescens Strain P3 Improve the Induction of Systemic Resistance in Tobacco Against Tobacco Necrosis Virus.

ABSTRACT Application of salicylic acid induces systemic acquired resistance in tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of tobacco, but did not improve the ability of CHA0 to induce systemic resistance in tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobacco in a gnotobiotic system.

The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element.

The estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.

Predictive value of readiness, importance, and confidence in ability to change drinking and smoking.

BACKGROUND: Visual analog scales (VAS) are sometimes used to assess change constructs that are often considered critical for change. Aims of Study: 1.) To determine the association of readiness to change, importance of changing and confidence in ability to change alcohol and tobacco use at baseline with the risk for drinking (more than 21 drinks per week/6 drinks or more on a single occasion more than once per month) and smoking (one or more cigarettes per day) six months later. 2.) To determine the association of readiness, importance and confidence with alcohol (number of drinks/week, number of binge drinking episodes/month) and tobacco (number of cigarettes/day) use at six months. METHODS: This is a secondary analysis of data from a multi-substance brief intervention randomized trial. A sample of 461 Swiss young men was analyzed as a prospective cohort. Participants were assessed at baseline and six months later on alcohol and tobacco use, and at baseline on readiness to change, importance of changing and confidence in ability to change constructs, using visual analog scales ranging from 1-10 for drinking and smoking behaviors. Regression models controlling for receipt of brief intervention were employed for each change construct. The lowest level (1-4) of each scale was the reference group that was compared to the medium (5-7) and high (8-10) levels. RESULTS: Among the 377 subjects reporting unhealthy alcohol use at baseline, mean (SD) readiness, importance and confidence to change drinking scores were 3.9 (3.0), 2.7 (2.2) and 7.2 (3.0), respectively. At follow-up, 108 (29%) reported no unhealthy alcohol use. Readiness was not associated with being risk-free at follow-up, but high importance (OR 2.94; 1.15, 7.50) and high confidence (OR 2.88; 1.46, 5.68) were. Among the 255 smokers at baseline, mean readiness, importance and confidence to change smoking scores were 4.6 (2.6), 5.3 (2.6) and 5.9 (2.7), respectively. At follow-up, 13% (33) reported no longer smoking. Neither readiness nor importance was associated with being a non-smoker, whereas high confidence (OR 3.29; 1.12, 9.62) was. CONCLUSIONS: High confidence in ability to change was associated with favorable outcomes for both drinking and smoking, whereas high importance was associated only with a favorable drinking outcome. This study points to the value of confidence as an important predictor of successful change for both drinking and smoking, and shows the value of importance in predicting successful changes in alcohol use. TRIAL REGISTRATION NUMBER: ISRCTN78822107.

Mécanismes moléculaires de la sécrétion d'insuline : implication de Slac2c/MyRIP, protéine effectrice de Rab27a, et rôle des phosphoinositides dans le processus d'exocytose

Résumé : La sécrétion de l'insuline en réponse au glucose circulant dans le sang est la fonction principale de la cellule β. La perte de cette fonction est une des caractéristiques du diabète de type 2. L'exocytose est une fonction cellulaire indispensable au renouvellement des composants lipidiques et protéiques de la membrane cellulaire, à la communication entre les cellules et au maintien d'un environnement adéquat. On peut distinguer deux types d'exocytose : l'exocytose constitutive et l'exocytose régulée. Cette dernière est déclenchée par des stimuli externes. L'exocytose régulée est contrôlée au niveau de la fusion des vésicules de sécrétion avec la membrane plasmique. Certains composants moléculaires impliqués dans ce processus font partie de la famille des GTPases Rab. Les deux membres de cette famille impliqués sont Rab3 et Rab27. Nous avons étudié le rôle de la GTPase Rab27 dans les cellules INS-1E, une lignée cellulaire pancréatique β qui sécrète de l'insuline de façon régulée. Nous avons trouvé que la diminution d'expression de la protéine en utilisant le technique de « RNA interference » diminue la sécrétion stimulée, mais que la distribution des granules n'est nullement affectées par ce changement d'activité intrinsèque. Un des effecteurs identifiés de cette GTPase est Slac2c/MyRIP. Cette protéine possède plusieurs domaines fonctionnels dont un qui lui permet de se lier à l'actine, constituant du cytosquelette cellulaire. L'ensemble de nos résultats suggèrent que Rab27 et MyRIP font partie d'un complexe permettant l'interaction de la granule de sécrétion avec le cytosquelette d'actine corticale et participent à la régulation des dernières étapes de l'exocytose d'insuline. Ensuite, nous avons étudié les phosphoinositides (PI). Les phosphoinositides sont d'importantes molécules impliquées dans le régulation du trafic vésiculaire. Nous avons trouvé que le phosphatidylinosito1-4-phosphate (PI4P) et le phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) augmentent la sécrétion sous l'action de 10µM de Ca2+ dans les cellules INS-1E perméabilisées avec la streptolysine-O. En plus, nous avons démontré que l'exocytose est diminuée dans les cellules intactes exprimant une protéine qui séquestre le PI(4,5)P2. Une diminution similaire est observée en diminuant l'expression de deux enzymes impliquées dans la production du PI(4,5)P2, la PI4Kinase β type III et la PIP5Kinase γ type I. Pour clarifier le mécanisme d'action des PI, nous avons investigué l'implication de trois cibles potentielles des PI, la PLD1, CAPS1 et Mint1. Pour ce faire, nous avons réduit le niveau d'expression endogène de ces protéines, ce qui inhibe la libération d'hormones provoquée par le glucose. Tout ceci indique donc que la production du PI(4,5)P2 est nécessaire pour le contrôle de la sécrétion et suggère qu'une partie de l'effet du PI sur la sécrétion pourrait être exercé par l'activation de la PLD1, CAPS1 et Mint1. Abstract Insulin release from pancreatic β-cells plays an essential role in the achievement of blood glucose homeostasis and defects in the regulation of this process lead to profound metabolic disorders and hyperglycaemia (eg. type 2 diabetes). Almost every cell in our organism releases proteins and other biological compounds using a fundamental cellular process known as constitutive exocytosis. In exocrine and endocrine glands, the cells are endowed with an additional and more refined release mechanism directly tuned by extracellular signals. This process, referred to as regulated exocytosis, ensures the timely delivery of molecules such as peptide hormones and digestive enzymes to match the moment¬-to-moment requirements of the organism. Some of the molecular components involved in this process have been identified, including Rab3 and Rab27, two GTPases that regulate the final steps of secretion in many cells. We investigated the involvement of Rab27 GTPase in the secretory process of the insulin-secreting cell line INS-1E. We found that selective reduction of Rab27 expression by RNA interference did not alter granule distribution but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors revealed that Slac2c/MyRIP is associated with granules and attenuation of Slac2c expression severely impaired hormone release. This protein contains several functional domains, including, a binding domain for the cellular cytoskeleton constituent actin. Taken together our data suggest the Rab27 and MyRIP are part of a complex mediating the interaction of secretory granules with cortical cytoskeleton and participate to the regulation of the final steps in insulin exoctytosis. In the second part of the thesis, we studied phosphoinositides (PI). Phosphoinositides are important molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinosito1-4-phosphate (PI4P) and phosphatidylinosito1-4,5-biphosphate (PI(4,5)P2) increase the secretory response triggered by 10µM Ca2+ in streptolysin-O permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P2. A similar decrease was observed after silencing of two enzymes involved in PI(4,5)P2 production, type III PI4Kinase β and type I PIP5Kinase γ, by RNA interference. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of exocytosis of three potential PI targets, PLD1, CAPS1 and Mint1. Transfection of cells with silencers capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose. Our data indicate that the production PI(4,5)P2 is necessary for proper control of p-cell secretion and suggest that at least part of the effects of PI on insulin exocytosis could be exerted through the activation of PLD1, CAPS1 and Mint1.

Self construction, cognitive conflicts and polarization in bulimia nervosa

This study explores the cognitive structures, understood as construct systems, of patients suffering from bulimia nervosa (BN). Previous studies investigated the construct systems of disordered eaters suggesting that they had a higher distance between their construction of the self and the «ideal self», and also more rigidity. In addition to these aspects, this study explored the presence of implicative dilemmas (ID). Thirty two women who met criteria for BN and were treated in a specialized center were compared to a non clinical group composed by 32 women matched by age. All participants were assessed using Repertory Grid Technique (RGT). In BN patients it was more common (71.9%) to find IDs than in controls (18.8%). They also showed higher polarization and higher self-ideal discrepancies (even more for those with a long history of BN). The measures provided by the RGT can be useful for the assessment of self-construction and cognitive conflicts in BN patients and to appreciate their role in this disorder. In addition, this technique could be helpful for clinicians to explore the patient"s constructs system, and specially to identify IDs that could be maintaining the symptoms or hindering change in order to focus on them to facilitate improvement.

Cuando Frankenstein encontró a Dorian Gray: dandysmo, post-identidad y sujetos virtuales

The appearance and popularization of the internet has created new forms of writing, which compel us to think anew about identity and subjectivity. Webjournals or blogs are specially interesting because they are a massive phenomenon that use autobiographical writing in a peculiar way. These forms of writing stress a particular paradox of the genre: the coexistence between a purpose of private, confessional and spontaneous writing and a public image, carefully built, as a result of its writing. The technology is new, but, in fact, the paradox is old. This paper tries to explore this old paradox, our eternal condition of cyborgs, our use of technologies in order to construct a public, unique and recognizably identity. In oder to do so, I will try to show the virtual condition of any written individual ¿this issue has already been dealt with by autobiographical studies¿, focusing on blogs, and especially on concrete example (Lord Whimsy¿s Journal). I will pay attention to gender as a technology that constructs identity and, at the same time, is deconstructed by the autobiographical narratives analyzed. In short, I attempt to show that virtual and autobiographical discourse do not bring forth a new kind of subject but the permanence of an old phenomenon "clearly developed by dandyism, for instance¿: the use of tehnologies to re-invent, re-formulate and re-construct us as multiple, hybrid and mixed subjects."

Unnatural Mothers, Mothering Unnaturally: Technologies of Reproduction and the Politics of Maternity in Hiromi Goto's "Hopeful Monsters"

Through scientific discourse and reproductive technologies, the reproductive body and the maternal body continue to be constructed as ‘natural’. At the same time,these technologies have begun to blur the boundaries between what is consideredan acceptable reproductive body, and consequently an acceptable maternal body,and an unnatural or a socially undesireable one. As science purports to offerwomen greater control over how and when they choose to procreate, through methods which range between delaying or eliminating the possibility of contraception to those which extend the possibility of conception to postmenopausal or infertile women, these same procedures raise questions about thenature and ‘naturalness’ of reproduction. Added to these concerns are thesuitablility of the reproductive body as a maternal body. Consequently, and moreand more frequently, bodies which defy ideals about maternity and motherhoodemerge, and questions about what it means to mother are raised. Bodies whichcontest the construction of motherhood as natural are frequently represented asmonstrous or freakish, and the debate between science and nature is heightened.Hiromi Goto’s short story ‘Hopeful Monsters’ resists the construction of the‘natural’ maternal body by highlighting the way in which women’s bodies areshaped by scientific discourse. In turn, images of ‘monstrous’ mothers emerge andare challenged, suggesting the need to reimagine what it means to mother and whatit means to be a mother. Through reading a selection of the stories this paper willinterrogate possible alternatives to constructions of the ‘natural’ maternal body and motherhood, suggesting that the Goto’s ‘monsters’ are perhaps only monstrous as a result of scientific discourse which constructs them as such.

Prospective association of the SHARE-operationalized frailty phenotype with adverse health outcomes: evidence from 60+ community-dwelling Europeans living in 11 countries.

BACKGROUND: Among the many definitions of frailty, the frailty phenotype defined by Fried et al. is one of few constructs that has been repeatedly validated: first in the Cardiovascular Health Study (CHS) and subsequently in other large cohorts in the North America. In Europe, the Survey of Health, Aging and Retirement in Europe (SHARE) is a gold mine of individual, economic and health information that can provide insight into better understanding of frailty across diverse population settings. A recent adaptation of the original five CHS-frailty criteria was proposed to make use of SHARE data and measure frailty in the European population. To test the validity of the SHARE operationalized frailty phenotype, this study aims to evaluate its prospective association with adverse health outcomes. METHODS: Data are from 11,015 community-dwelling men and women aged 60+ participating in wave 1 and 2 of the Survey of Health, Aging and Retirement in Europe, a population-based survey. Multivariate logistic regression analyses were used to assess the 2-year follow up effect of SHARE-operationalized frailty phenotype on the incidence of disability (disability-free at baseline) and on worsening disability and morbidity, adjusting for age, sex, income and baseline morbidity and disability. RESULTS: At 2-year follow up, frail individuals were at increased risk for: developing mobility (OR 3.07, 95% CI, 1.02-9.36), IADL (OR 5.52, 95% CI, 3.76-8.10) and BADL (OR 5.13, 95% CI, 3.53-7.44) disability; worsening mobility (OR 2.94, 95% CI, 2.19- 3.93) IADL (OR 4.43, 95% CI, 3.19-6.15) and BADL disability (OR 4.53, 95% CI, 3.14-6.54); and worsening morbidity (OR 1.77, 95% CI, 1.35-2.32). These associations were significant even among the prefrail, but with a lower magnitude of effect. CONCLUSIONS: The SHARE-operationalized frailty phenotype is significantly associated with all tested health outcomes independent of baseline morbidity and disability in community-dwelling men and women aged 60 and older living in Europe. The robustness of results validate the use of this phenotype in the SHARE survey for future research on frailty in Europe.

Clinton, Iowa: Railroad Town, 2003

Clinton, Iowa, one of the first railroad crossings over the Mississippi River, has been a major gateway to the Great Plains and beyond since 1859. For more than 100 years, the railroads employed thousands and supported a good quality of life in Clinton. Railroad activities peaked both nationally and in Clinton during and after World War II. By the 1990s, the Union Pacific was redeveloping their railroad facilities adjacent to Camanche Avenue and U.S. Highway 30. The legacy of the railroad in Clinton has been preserved in this study.

Virtuaalikirjaston rooli tutkijoiden tiedonhankintakäytännöissä

Summary: The role of subject gateway libraries in researchers' information seeking