Kinetic and Structural Studies on Flavin-dependent Enzymes involved in Glycine Betaine Biosynthesis and Propionate 3-nitronate Detoxification

Flavin-dependent enzymes are characterized by an amazing chemical versatility and play important roles in different cellular pathways. The FAD-containing choline oxidase from Arthrobacter globiformis oxidizes choline to glycine betaine and retains the intermediate betaine aldehyde in the active site. The reduced FAD is oxidized by oxygen. Glycine betaine is an important osmoprotectant accumulated by bacteria, plants, and animals in response to stress conditions. The FMN-containing nitronate monooxygenase detoxifies the deadly toxin propionate 3-nitronate which is produced by plants and fungi as defense mechanism against herbivores. The catalytic mechanism of fungal nitronate monooxygenase (NMO) was characterized, but little is known about bacterial NMOs. In this dissertation the crystal structure of choline oxidase in complex with glycine betaine was solved and the roles of the residue F357 in the oxidative half reaction investigated by combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects and molecular dynamics simulations. Expression trials of human choline dehydrogenase were carried out and a homology model based on choline oxidase was generated. A bacterial nitronate monooxygenase from Pseudomonas aeruginosa PAO1 Pa-NMO was kinetically and structurally characterized and four conserved motifs were described that identify Class 1 NMO. Two hypothetical NMOs from P. aeruginosa PAO1 and one from Helicobacter pylori not carrying the motifs of Class 1 NMO were cloned and tested for nitronate monooxygenase activity. The crystal structure of choline oxidase in complex with glycine betaine highlighted two different conformations of loop 250-255 at the dimer interface that is proposed to control substrate access to the active site. The side chain of F357 was associated with a slow isomerization in the oxidation of the reduced FAD of choline oxidase. The first crystal structure of an NMO highlighted active site residues for site-directed mutagenesis studies. The gene function prediction for NMO was improved with the four conserved motifs of Pa-NMO. The two hypothetical NMOs from P. aeruginosa PAO1 were shown to possess a different enzymatic activity and to identify two distinct classes of enzymes. The hypothetical NMO from H. pilory did not exhibit NMO activity and contained iron as cofactor

Of bellies and books: (re)positioning the subject within the education/pregnancy nexus in Mozambique

‘Of Bellies and Books’ refers to pregnancy and formal education, constructed as mutually exclusive processes. This thesis explores that opposition by tracing the confluence of discourses through which it is produced. In so doing, it dissolves dichotomies and proposes a shift to the subject as both constituted by and constituting of discourses. Both academic research and global and national social policy construct teenage pregnancy as problematic. This is heightened in development contexts, where in-school pregnancy triggers Malthusian fears of overpopulation and consequential poverty increase. Conversely, formal education and training are represented as a means to personal development and success, through acquiring knowledge and skills leading to formal employment and individual empowerment. In this sense, schooling is constructed as a symbol of - or entrance to - modernity, while pregnancy and parenthood are defined in terms of the opportunities they prevent. From this perspective, in-school pregnancy works against individual and social progress and is synonymous with backwardness and tradition within a modernising and globalised world. Exploring in-school pregnancy in this thesis becomes a means through which to revoke the binary symbolised by tradition and modernity which produces a deficit view of the pregnant schoolgirl. Within this context, the study has been driven by the following research questions: · How do education policy and practice frame in-school pregnancy in Mozambique? · How do families interpret and regulate in-school pregnancy? · How do young people – young women – navigate the available discourses in the performance of their identities? Stimulated by a desire to explore the national policy tackling in-school pregnancy indicating that pregnant schoolgirls should be transferred to night courses, the empirical data collection took place within 10 months in and around the capital Maputo. It entailed documentary analysis, interviews with 10 Ministry of Education officials, 20 school teachers and 33 young people (25 girls and 8 men/boys) in and out of education. Through the generation and analysis of data, I develop a nuanced interpretation of the discourses that construct and regulate in-school pregnancy within schools and families. Within the institutional space of schools, a textual analysis of the policy shows how language borrowed from the biomedical and legal fields is directed towards the production of in-school pregnancy as unwanted, unplanned and ultimately ‘wrong’. This normalises the difference between pregnant and non-pregnant schoolgirls, producing transfer to night courses as a rational strategy to tackle in-school pregnancy. Although understood as a means to bridge the gender gap in education by tackling one of the main causes of female dropout, the current policy acts de facto as a highway to dropout, thereby reproducing gender exclusion. Within families, pregnancy initiates the complex procedure of family formation by drawing on the mutually exclusive categories of childhood and adulthood and symbolising the transition between the two. I contend that these two spaces, schools and families, often associated by research participants with modernity and tradition respectively, are not stable and homogenous constructs, but offer shifting and contingent sets of norms which are both conflicting and intersecting. By engaging with young people's narratives, I argue that pregnant schoolgirls, while being constructed by discursive norms, also resist and react to them. At school, young pregnant females enact a number of strategies to resist transfer to night courses. At home, they resist family formation and find ways to combine their multiple identities. By drawing on this, I ultimately contend that young pregnant schoolgirls navigate different regulatory frameworks in the production of their identities. This means that the itineraries they construct in crossing boundaries within normative frameworks constitute their identities and reposition them as travellers

"Branch and Bound" and "Branch and Reduce" approaches for a class of D.C. Programs

In this Ph.D. thesis an important class of d.c. programs is studied from both a theoretical and an algorithmic point of view. This class of problems is computationally studied with a "branch and bound" and a "branch and reduce" approaches, pointing out the effectiveness of partitioning rules and of stack policies for managing the branches. In this light, the results appeared in the literature are extended and deepened on. This study can be virtually divided into three main steps

Pedagogy, curriculum, teaching practices and teacher education in developing countries

This rigorous literature review focused on pedagogy, curriculum, teaching practices and teacher education in developing countries. It aimed to: 1. review existing evidence on the review topic to inform programme design and policy making undertaken by the DFID, other agencies and researchers 2. identify critical evidence gaps to guide the development of future research programme

Dynamic changes in synaptic plasticity genes in ipsilateral and contralateral inferior colliculus following unilateral noise-induced hearing loss

Unilateral noise-induced hearing loss reduces the input to the central auditory pathway disrupting the excitatory and inhibitory inputs to the inferior colliculus (IC), an important binaural processing center. Little is known about the compensatory synaptic changes that occur in the IC as a consequence of unilateral noise-induced hearing loss. To address this issue, Sprague–Dawley rats underwent unilateral noise exposure resulting in severe unilateral hearing loss. IC tissues from the contralateral and ipsilateral IC were evaluated for acute (2-d) and chronic (28-d) changes in the expression of 84 synaptic plasticity genes on a PCR array. Arc and Egr1 genes were further visualized by in situ hybridization to validate the PCR results. None of the genes were upregulated, but many were downregulated post-exposure. At 2-d post-exposure, more than 75% of the genes were significantly downregulated in the contralateral IC, while only two were downregulated in the ipsilateral IC. Many of the downregulated genes were related to long-term depression, long-term potentiation, cell adhesion, immediate early genes, neural receptors and postsynaptic density. At 28-d post-exposure, the gene expression pattern was reversed with more than 85% of genes in the ipsilateral IC now downregulated. Most genes previously downregulated in the contralateral IC 2-d post-exposure had recovered; less than 15% remained downregulated. These time-dependent, asymmetric changes in synaptic plasticity gene expression could shed new light on the perceptual deficits associated with unilateral hearing loss and the dynamic structural and functional changes that occur in the IC days and months following unilateral noise-induced hearing loss

Effect of biofilm removal from the occlusal tooth surfaces on fluorescence measurements. A clinical study

Aim: Early diagnosis and monitoring of caries lesions are the most important issues of primary and secondary prevention policies.The intraoral VistaCamiX(DurrDental, Bietigheim‐Bissingen,Germany) uses the fluorescence phenomenon for a non‐invasive, quantitative caries diagnosis. In order to make a precise evaluation the tooth surface must be completely cleaned and without biofilm. The current study aimed to evaluate the effects of biofilm removal, using air‐polishing device (Combi,MectronSpA) with glycine, on fluorescence VistaCam iX camera quantitative measurements of caries. Methods: Patients with complete permanent dentition without any kind of restorative treatments in the lateral and posterior section of upper and lower dental arches were enrolled. The occlusal surfaces of molars and premolars were photographed using the fluorescence terminal Proof of the intraoral camera VistaCam iX before and after air polishing glycine procedures, registering the highest value gained for each occlusal surface. Results:133cuspidate permanent teeth of patients aged between 13 and 25 were analyzed. Descriptive analysis showed an average of 0.82 (SD=0.65; Min=0.00; Max=1.80; Median=1.20) and of 0.93 (SD=0.60; Min=0.00; Max=1.70; Median=1.20) for values before and after treatment, respectively.The scores assigned by VistaCam iX Proof fluorescence based camera to the occlusal surfaces, after the air‐polishing treatment, are averagely higher than those before treatment, especially in the diagnosis of initial tooth decay. Conclusion: Biofilm removal with glycine air‐polishing improves the VistaCam camera accuracy in recognizing healthy tissue from the decayed one, due to the fact that air-polishing treatment increases the decayed tissue reaction to the fluorescence

Effect of Agronomic Techniques on Aroma Composition of White Grapevines: A Review

Climate change with rising temperatures and the unpredictability of rainy events during ripening leads to tough challenges for the winemakers in preserving the quality of white grape varieties. Grape quality is a complex concept that mainly refers to berry chemical composition, including secondary metabolites such as aroma compounds that in white berries play a key role in the identity of the wine. Terpenes, thiols, C13-norisoprenoids, methoxypyrazines, and nonterpenic alcohols are the most important aroma compounds in white grapes and several of them can be found as free volatiles or bound as glycoconjugate molecules. Agronomic practices in vineyards, such as biostimulant application, irrigation, defoliation, training systems, foliar fertilization, and bunch thinning, can have a positive effect on their concentration. This review aims to highlight the aromatic characteristics of the most utilized white grape cultivars and focus on agronomic techniques in the vineyard to enhance, implement, and intensify their aromatic characteristics. The purpose of this review paper is therefore to investigate the state of the art regarding the exaltation of aroma in white grapes in the scientific literature through sustainable agronomic techniques, but further future investigations are considered necessary given the uncertainty concerning some mechanisms of biosynthesis and their correlation with such agronomic practices

Haemodynamic Adaptive Mechanisms at High Altitude: Comparison between European Lowlanders and Nepalese Highlanders

Background: Exposure to high altitudes determines several adaptive mechanisms affecting in a complex way the whole cardiovascular, respiratory, endocrine systems because of the hypobaric hypoxic condition. The aim of our study was to evaluate the circulatory adaptive mechanisms at high altitudes, during a scientific expedition in the Himalayas. Methods: Arterial distensibility was assessed measuring carotid-radial and carotid-femoral pulse wave velocity. Tests were carried out at several altitudes, from 1350 to 5050 m above sea level, on 8 lowlander European researchers and 11 highlander Nepalese porters. Results: In Europeans, systolic blood pressure and pulse pressure increased slightly but significantly with altitude (p < 0.05 and p < 0.001, respectively). Norepinephrine showed a significant increase after the lowlanders had spent some time at high altitude (p < 0.001). With increasing altitude, a progressive increase in carotid-radial and carotid-femoral pulse wave velocity values was observed in lowlanders, showing a particularly significant increase (p < 0.001) after staying at high altitude (carotid-radial pulse wave velocity, median value (interquartile range) from 9.2 (7.9-10.0) to 11.2 (10.9-11.8) m/s and carotid-femoral pulse wave velocity from 8.5 (7.9-9.0) to 11.3 (10.9-11.8) m/s). At high altitudes (3400 and 5050 m above sea level), no significant differences were observed between highlanders and lowlanders in hemodynamic parameters (blood pressure, carotid-radial and carotid-femoral pulse wave velocity). Conclusions: The progressive arterial stiffening with altitude observed in European lowlanders could explain the increase in systolic and pulse pressure values observed at high altitudes in this ethnic group. Further studies are needed to evaluate the role of aortic stiffening in the pathogenesis of acute mountain sickness