Evaluation of antiradical assays used in determining the antioxidant capacity of pure compounds and plant extracts

The efficiency of the chemiluminescence luminol method and colorimetric DPPH and ABTS methods in evaluating the antiradical capacity of pure compounds and plant extracts with antioxidant potential is compared. In case of pure compounds, the values of parameter 'n' (number of radicals quenched per molecule of antiradical) for ascorbic acid, p-hydroquinone, catechol, quercetin, and rutin are similar when measured by colorimetric assays; however, considerably lower values of n are obtained with the luminol assay. The antiradical activity of extracts from male and female individuals of Baccharis burchelli and Baccharis crispa were determined by the luminol assay and expressed using the new Trolox® percentage (%Trolox®) parameter.

Green tea in transdermal formulation: HPLC method for quality control and in vitro drug release assays

RP-HPLC based analytical method for use in both quality control of green tea in a semisolid formulation and for in vitro drug release assays was developed and validated. The method was precise (CV < 5%), accurate (recovery between 98% and 102%), linear (R² > 0.99), robust, and specific for the determination of epigallocatechin 3-gallate (EGCG), caffeine (CAF), and gallic acid (GA). In a diffusion cell chamber, the release rate of EGCG was 8896.01 µg cm-2. This data showed that EGCG will be able to exert its systemic activity when delivered though the transdermal formulation, due to its good flux rates with the synthetic membrane.

THE APPLICATION OF AUTOMATED CORRELATION OPTIMIZED WARPING TO THE QUALITY EVALUATION OF Radix Puerariae thomsonii: CORRECTING RETENTION TIME SHIFT IN THE CHROMATOGRAPHIC FINGERPRINTS

The application of automated correlation optimized warping (ACOW) to the correction of retention time shift in the chromatographic fingerprints of Radix Puerariae thomsonii (RPT) was investigated. Twenty-seven samples were extracted from 9 batches of RPT products. The fingerprints of the 27 samples were established by the HPLC method. Because there is a retention time shift in the established fingerprints, the quality of these samples cannot be correctly evaluated by using similarity estimation and principal component analysis (PCA). Thus, the ACOW method was used to align these fingerprints. In the ACOW procedure, the warping parameters, which have a significant influence on the alignment result, were optimized by an automated algorithm. After correcting the retention time shift, the quality of these RPT samples was correctly evaluated by similarity estimation and PCA. It is demonstrated that ACOW is a practical method for aligning the chromatographic fingerprints of RPT. The combination of ACOW, similarity estimation, and PCA is shown to be a promising method for evaluating the quality of Traditional Chinese Medicine.

Sensitive Troponin Assays: Diagnostic and Prognostic Use in Cardiology

Cardiac troponins (cTns) are the recommended biochemical markers in the diagnosis of myocardial infarction (MI). They are very sensitive and tissue-specific but are limited by their delayed appearance in the circulation. Biochemical markers with more rapid release kinetics, e.g. myoglobin and especially heart-type fatty acid-binding protein (H-FABP), have been used to enhance the early identification of MI. The implementation of cTns into clinical practice has shown that cardiomyocyte injury occurs in many other clinical conditions than MI. The aim of this study was to evaluate the impact of modern and highly sensitive cTnI assays on the early diagnosis of MI. In a patient cohort with suspected MI, such a sensitive cTnI assay enhanced the early diagnostic accuracy when compared to a less sensitive cTnI assay and to myoglobin. When compared to H-FABP during the early hours after symptom onset, the sensitive cTnI assay showed at least similar and, after 6 hours, superior diagnostic accuracy. A positive cTnI test result had superior prognostic value when compared to H-FABP, even among early presenters. The prognostic value of cTn in acute heart failure (AHF) was evaluated in 364 patients who participated in the FINN-AKVA study. The patients presented with AHF but no acute coronary syndrome (ACS). Up to half of the patients had elevated cTn levels which were associated with higher 6-month mortality. The magnitude of cTn elevation was directly proportional to mortality. Finally, the clinical spectrum of cTnI elevations was evaluated in 991 cTnI positive emergency department (ED) patients. 83% of the patients had MI and 17% had cTnI elevation due to other clinical conditions. The latter patient group was characterized by lower absolute cTnI levels and – importantly – higher in-hospital mortality when compared to the MI patients. In conclusion, the use of a highly sensitive cTnI assay enhances the early diagnostic accuracy and risk stratification in suspected MI patients. Cardiac troponin elevations are highly prevalent also in other acute clinical conditions and indicate an adverse outcome of these patients.

Differentiation of Colletotrichum gloeosporioides isolates by using total proteins and esterase electrophoretic patterns and extracellular enzymes production

Isolates of Colletotrichum gloeosporioides (ISO-1, ISO-2, ISO-3, ISO-4, ISO-5 and ISO-6), the causal agent of anthracnose disease on mango fruits, were characterized by electrophoretic patterns of total proteins and esterase in polyacrylamida gel, and also, by production of extracellular enzymes on specific solid substrate. The electrophoretic analysis showed variation in number, intensity of coloration and position of the bands in the gel at each studied system tested. In contrast to the monomorphic behavior to total proteins, high esterase polymorfism was observed indicating difference among isolates. All isolates showed the activity of extracellular enzymes such as amylase, lipase, and protease with some variation among them. The proteolitic activity seemed to be more accentuated than the two other enzymes studied.

Conference Proceedings. Academic Mobility Blending Perspectives - Mobilité Académique Perspectives croisées

Les 21, 22 et 23 septembre 2006, le Département d’Études Françaises de l’Université de Turku (Finlande) a organisé une conférence internationale et bilingue (anglais et français) sur le thème de la mobilité académique ; le but de cette rencontre était de rendre possible la tenue d’un forum international et multidisciplinaire, susceptible d’être le siège de divers débats entre les différents acteurs de la mobilité académique (c’estàdire des étudiants, des chercheurs, des personnels enseignants et administratifs, etc.). Ainsi, ont été mis à contribution plus de cinquante intervenants, (tous issus de domaines aussi variés que la linguistique, les sciences de l’éducation, la didactique, l’anthropologie, la sociologie, la psychologie, l’histoire, la géographie, etc.) ainsi que cinq intervenants renommés1. La plupart des thèmes traités durant la conférence couvraient les champs suivants : l’organisation de la mobilité, les obstacles rencontrés par les candidats à la mobilité, l’intégration des étudiants en situation d’échange, le développement des programmes d’études, la mobilité virtuelle, l’apprentissage et l’enseignement des langues, la prise de cosncience interculturelle, le développement des compétences, la perception du système de mobilité académique et ses impacts sur la mobilité effective. L’intérêt du travail réalisé durant la conférence réside notamment dans le fait qu’il ne concentre pas uniquement des perspectives d’étudiants internationaux et en situation d’échange (comme c’est le cas de la plupart des travaux de recherche déjà menés sur ce sujet), mais aussi ceux d’autres corps : enseignants, chercheurs, etc. La contribution suivante contient un premier corpus de dixsept articles, répartis en trois sections : 1. Impacts de la mobilité étudiante ; 2. Formation en langues ; 3. Amélioration de la mobilité académique. À l’image de la conférence, la production qui suit est bilingue : huit des articles sont rédigés en français, et les neuf autres en anglais. Certains auteurs n’ont pas pu assister à la conférence mais ont tout de même souhaité apparaître dans cet ouvrage. Dans la première section de l’ouvrage, Sandrine Billaud tâche de mettre à jour les principaux obstacles à la mobilité étudiante en France (logement, organisation des universités, démarches administratives), et propose à ce sujet quelques pistes d’amélioration. Vient ensuite un article de Dominique Ulma, laquelle se penche sur la mobilité académique régnant au sein des Instituts Universitaires de Formation des Maîtres (IUFM) ; elle s’est tout particulièrement concentrée sur l’enthousiasme des stagiaires visàvis de la mobilité, et sur les bénéfices qu’apporte la mobilité Erasmus à ce type précis d’étudiant. Ensuite, dans un troisième article, Magali Hardoin s’interroge sur les potentialités éducationnelles de la mobilité des enseignantsstagiaires, et tâche de définir l’impact de celleci sur la construction de leur profil professionnel. Après cela arrive un groupe de trois articles, tous réalisés à bases d’observations faites dans l’enseignement supérieur espagnol, et qui traitent respectivement de la portée qu’a le programme de triple formation en langues européennes appliquées pour les étudiants en mobilité (Marián MorónMartín), des conséquences qu’occasionne la présence d’étudiants étrangers dans les classes de traductions (Dimitra Tsokaktsidu), et des réalités de l’intégration sur un campus espagnol d’étudiants américains en situation d’échange (Guadalupe Soriano Barabino). Le dernier article de la section, issu d’une étude sur la situation dans les institutions japonaises, fait état de la situation des programmes de doubles diplômes existant entre des établissements japonais et étrangers, et tente de voir quel est l’impact exact de tels programmes pour les institutions japonaises (Mihoko Teshigawara, Riichi Murakami and Yoneo Yano). La seconde section est elle consacrée à la relation entre apprentissage et enseignement des langues et mobilité académique. Dans un premier article, Martine Eisenbeis s’intéresse à des modules multimédia réalisés à base du film « L’auberge espagnole », de Cédric Klapish (2001), et destinés aux étudiants en mobilité désireux d’apprendre et/ou améliorer leur français par des méthodes moins classiques. Viennent ensuite les articles de Jeanine Gerbault et Sabine Ylönen, lesquels traitent d’un projet européen visant à supporter la mobilité étudiante par la création d’un programme multimédia de formation linguistique et culturelle pour les étudiants en situation de mobilité (le nom du projet est EUROMOBIL). Ensuite, un article de Pascal Schaller s’intéresse aux différents types d’activités que les étudiants en séjour à l’étranger expérimentent dans le cadre de leur formation en langue. Enfin, la section s’achève avec une contribution de Patricia KohlerBally, consacrée à un programme bilingue coordonné par l’Université de Fribourg (Suisse). La troisième et dernière section propose quelques pistes de réflexion destinées à améliorer la mobilité académique des étudiants et des enseignants ; dans ce cadre seront donc évoquées les questions de l’égalité face à la mobilité étudiante, de la préparation nécessitée par celleci, et de la prise de conscience interculturelle. Dans un premier chapitre, Javier Mato et Bego

Upconverting Phosphor Technology: Exceptional Photoluminescent Properties Light Up Homogeneous Bioanalytical Assays

The aim of the present study was to demonstrate the wide applicability of the novel photoluminescent labels called upconverting phosphors (UCPs) in proximity-based bioanalytical assays. The exceptional features of the lanthanide-doped inorganic UCP compounds stem from their capability for photon upconversion resulting in anti-Stokes photoluminescence at visible wavelengths under near-infrared (NIR) excitation. Major limitations related to conventional photoluminescent labels are avoided, rendering the UCPs a competitive next-generation label technology. First, the background luminescence is minimized due to total elimination of autofluorescence. Consequently, improvements in detectability are expected. Second, at the long wavelengths (>600 nm) used for exciting and detecting the UCPs, the transmittance of sample matrixes is significantly greater in comparison with shorter wavelengths. Colored samples are no longer an obstacle to the luminescence measurement, and more flexibility is allowed even in homogeneous assay concepts, where the sample matrix remains present during the entire analysis procedure, including label detection. To transform a UCP particle into a biocompatible label suitable for bioanalytical assays, it must be colloidal in an aqueous environment and covered with biomolecules capable of recognizing the analyte molecule. At the beginning of this study, only UCP bulk material was available, and it was necessary to process the material to submicrometer-sized particles prior to use. Later, the ground UCPs, with irregular shape, wide size-distribution and heterogeneous luminescence properties, were substituted by a smaller-sized spherical UCP material. The surface functionalization of the UCPs was realized by producing a thin hydrophilic coating. Polymer adsorption on the UCP surface is a simple way to introduce functional groups for bioconjugation purposes, but possible stability issues encouraged us to optimize an optional silica-encapsulation method which produces a coating that is not detached in storage or assay conditions. An extremely thin monolayer around the UCPs was pursued due to their intended use as short-distance energy donors, and much attention was paid to controlling the thickness of the coating. The performance of the UCP technology was evaluated in three different homogeneous resonance energy transfer-based bioanalytical assays: a competitive ligand binding assay, a hybridization assay for nucleic acid detection and an enzyme activity assay. To complete the list, a competitive immunoassay has been published previously. Our systematic investigation showed that a nonradiative energy transfer mechanism is indeed involved, when a UCP and an acceptor fluorophore are brought into close proximity in aqueous suspension. This process is the basis for the above-mentioned homogeneous assays, in which the distance between the fluorescent species depends on a specific biomolecular binding event. According to the studies, the submicrometer-sized UCP labels allow versatile proximity-based bioanalysis with low detection limits (a low-nanomolar concentration for biotin, 0.01 U for benzonase enzyme, 0.35 nM for target DNA sequence).

Simulation methods in the modelling of bioaffinity assays

Computational model-based simulation methods were developed for the modelling of bioaffinity assays. Bioaffinity-based methods are widely used to quantify a biological substance in biological research, development and in routine clinical in vitro diagnostics. Bioaffinity assays are based on the high affinity and structural specificity between the binding biomolecules. The simulation methods developed are based on the mechanistic assay model, which relies on the chemical reaction kinetics and describes the forming of a bound component as a function of time from the initial binding interaction. The simulation methods were focused on studying the behaviour and the reliability of bioaffinity assay and the possibilities the modelling methods of binding reaction kinetics provide, such as predicting assay results even before the binding reaction has reached equilibrium. For example, a rapid quantitative result from a clinical bioaffinity assay sample can be very significant, e.g. even the smallest elevation of a heart muscle marker reveals a cardiac injury. The simulation methods were used to identify critical error factors in rapid bioaffinity assays. A new kinetic calibration method was developed to calibrate a measurement system by kinetic measurement data utilizing only one standard concentration. A nodebased method was developed to model multi-component binding reactions, which have been a challenge to traditional numerical methods. The node-method was also used to model protein adsorption as an example of nonspecific binding of biomolecules. These methods have been compared with the experimental data from practice and can be utilized in in vitro diagnostics, drug discovery and in medical imaging.

A platform for anti-biofilm assays combining viability, biomass and matrix quantifications in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Bacteria can exist as planktonic, the lifestyle in which single cells exist in suspension, and as biofilms, which are surface-attached bacterial communities embedded in a selfproduced matrix. Most of the antibiotics and the methods for antimicrobial work have been developed for planktonic bacteria. However, the majority of the bacteria in natural habitats live as biofilms. Biofilms develop dauntingly fast high resistance towards conventional antibacterial treatments and thus, there is a great need to meet the demands of effective anti-biofilm therapy. In this thesis project it was attempted to fill the void of anti-biofilm screening methods by developing a platform of assays that evaluate the effect that screened compounds have on the total biomass, viability and the extracellular polysaccharide (EPS) layer of the biofilms. Additionally, a new method for studying biofilms and their interactions with compounds in a continuous flow system was developed using capillary electrochromatography (CEC). The screening platform was utilized with a screening campaign using a small library of cinchona alkaloids. The assays were optimized to be statistically robust enough for screening. The first assay, based on crystal violet staining, measures total biofilm biomass, and it was automated using a liquid handling workstation to decrease the manual workload and signal variation. The second assay, based on resazurin staining, measures viability of the biofilm, and it was thoroughly optimized for the strain used, but was then a very simple and fast method to be used for primary screening. The fluorescent resazurin probe is not toxic to the biofilms. In fact, it was also shown in this project that staining the biofilms with resazurin prior to staining with crystal violet had no effect on the latter and they can be used in sequence on the same screening plate. This sequential addition step was indeed a major improvement on the use of reagents and consumables and also shortened the work time. As a third assay in the platform a wheat germ agglutinin based assay was added to evaluate the effect a compound has on the EPS layer. Using this assay it was found that even if compounds might have clear effect on both biomass and viability, the EPS layer can be left untouched or even be increased. This is a clear implication of the importance of using several assays to be able to find “true hits” in a screening setting. In the pilot study of screening for antimicrobial and anti-biofilm effects using a cinchona alkaloid library, one compound was found to have antimicrobial effect against planktonic bacteria and prevent biofilm formation at low micromolar concentration. To eradicate biofilms, a higher concentration was needed. It was also shown that the chemical space occupied by the active compound was slightly different than the rest of the cinchona alkaloids as well as the rest of the compounds used for validatory screening during the optimization processes of the separate assays.

Application of capillary electrophoretic methods for determining carbohydrates and aliphatic carboxylic acids formed during wood processing

Knowledge of the behaviour of cellulose, hemicelluloses, and lignin during wood and pulp processing is essential for understanding and controlling the processes. Determination of monosaccharide composition gives information about the structural polysaccharide composition of wood material and helps when determining the quality of fibrous products. In addition, monitoring of the acidic degradation products gives information of the extent of degradation of lignin and polysaccharides. This work describes two capillary electrophoretic methods developed for the analysis of monosaccharides and for the determination of aliphatic carboxylic acids from alkaline oxidation solutions of lignin and wood. Capillary electrophoresis (CE), in its many variants is an alternative separation technique to chromatographic methods. In capillary zone electrophoresis (CZE) the fused silica capillary is filled with an electrolyte solution. An applied voltage generates a field across the capillary. The movement of the ions under electric field is based on the charge and hydrodynamic radius of ions. Carbohydrates contain hydroxyl groups that are ionised only in strongly alkaline conditions. After ionisation, the structures are suitable for electrophoretic analysis and identification through either indirect UV detection or electrochemical detection. The current work presents a new capillary zone electrophoretic method, relying on in-capillary reaction and direct UV detection at the wavelength of 270 nm. The method has been used for the simultaneous separation of neutral carbohydrates, including mono- and disaccharides and sugar alcohols. The in-capillary reaction produces negatively charged and UV-absorbing compounds. The optimised method was applied to real samples. The methodology is fast since no other sample preparation, except dilution, is required. A new method for aliphatic carboxylic acids in highly alkaline process liquids was developed. The goal was to develop a method for the simultaneous analysis of the dicarboxylic acids, hydroxy acids and volatile acids that are oxidation and degradation products of lignin and wood polysaccharides. The CZE method was applied to three process cases. First, the fate of lignin under alkaline oxidation conditions was monitored by determining the level of carboxylic acids from process solutions. In the second application, the degradation of spruce wood using alkaline and catalysed alkaline oxidation were compared by determining carboxylic acids from the process solutions. In addition, the effectiveness of membrane filtration and preparative liquid chromatography in the enrichment of hydroxy acids from black liquor was evaluated, by analysing the effluents with capillary electrophoresis.

Determination of chlorophenols from water by solid phase microextraction - ion mobility spectrometry (SPME-IMS)

Chlorophenols have been classified as possible carcinogens for humans. Chlorophenols have been used as pesticides and wood preservatives. In Finland, during 1930 – 1980s, saw mills used KY-5 wood preservative that contained 2,4,6-TCP, 2,3,4,6-TeCP and PCP. Especially in Finland chlorophenols have entered the environment by leaking from contaminated grounds of old saw mills. Although chlorophenol concentrations found in environment do not cause acute concern, long term exposure can increase the risk of cancer. SPME is relatively cheap and simple sampling method, in which the sample extraction and concentration are performed in a single step. Solvents are not required in SPME. IMS is based on the detection of sample ion drift times. Based on the drift times, reduced mobilities are calculated, which are comparable despite the measurement conditions. SPME-IMS coupling has not been used earlier in the determination of chlorophenols from water samples. The scope of this work was to study, if SPME-IMS system is suitable for detecting chloro-phenols from water samples. The aim was to determine the most optimal extraction condi-tions, which were then applied to real water samples. Following detection limits were deter-mined: 2,4,6-TCP: 0.33 mg/l; 2,3,4,6-TeCP: 0.63 mg/l and PCP: 1.63 mg/l. Detection limits were high compared to the highest possible chlorophenol concentration that is allowed in Finnish drinking water, 10 μg/l. Detected concentrations from water sample differed from verified concentrations in the case of 2,3,4,6-TeCP by 4.6 % and in the case of 2,4,6-TCP by 48.4 %. Based on the results it can be said that SPME-IMS setup is suitable for preliminary analysis of mg/l chlorophenol concentrations from water samples.

Adult education policy in the European Union during the Lisbon decade : the shift to vocational adult learning and work skills matching

This doctoral dissertation investigates the adult education policy of the European Union (EU) in the framework of the Lisbon agenda 2000–2010, with a particular focus on the changes of policy orientation that occurred during this reference decade. The year 2006 can be considered, in fact, a turning point for the EU policy-making in the adult learning sector: a radical shift from a wide--ranging and comprehensive conception of educating adults towards a vocationally oriented understanding of this field and policy area has been observed, in particular in the second half of the so--called ‘Lisbon decade’. In this light, one of the principal objectives of the mainstream policy set by the Lisbon Strategy, that of fostering all forms of participation of adults in lifelong learning paths, appears to have muted its political background and vision in a very short period of time, reflecting an underlying polarisation and progressive transformation of European policy orientations. Hence, by means of content analysis and process tracing, it is shown that the new target of the EU adult education policy, in this framework, has shifted from citizens to workers, and the competence development model, borrowed from the corporate sector, has been established as the reference for the new policy road maps. This study draws on the theory of governance architectures and applies a post-ontological perspective to discuss whether the above trends are intrinsically due to the nature of the Lisbon Strategy, which encompasses education policies, and to what extent supranational actors and phenomena such as globalisation influence the European governance and decision--making. Moreover, it is shown that the way in which the EU is shaping the upgrading of skills and competences of adult learners is modeled around the needs of the ‘knowledge economy’, thus according a great deal of importance to the ‘new skills for new jobs’ and perhaps not enough to life skills in its broader sense which include, for example, social and civic competences: these are actually often promoted but rarely implemented in depth in the EU policy documents. In this framework, it is conveyed how different EU policy areas are intertwined and interrelated with global phenomena, and it is emphasised how far the building of the EU education systems should play a crucial role in the formation of critical thinking, civic competences and skills for a sustainable democratic citizenship, from which a truly cohesive and inclusive society fundamentally depend, and a model of environmental and cosmopolitan adult education is proposed in order to address the challenges of the new millennium. In conclusion, an appraisal of the EU’s public policy, along with some personal thoughts on how progress might be pursued and actualised, is outlined.

Response of Grilage-Like Structures by the Mobility Approach

This work presents an application of the Mobility Approach to the analysis of the power flow through grillage-like structures. Such structures are usually found in offshore platforms, supporting large and heavy machines. Different wave kinds (longitudinal, flexural and torsional) were initially considered in the power flow analysis between two beams joined in L. Beams excited by an in-plane point force showed strong coupling between longitudinal-flexural waves, while that for out-of-plane point force excitation, flexural-torsional waves coupling represents the most important mechanism of energy transmission. The response determination of grillages by the mobility approach requires the structure to be separated into simple beam-like structural components. Equations for rotations and displacements at the joints of all beams are written for as mobility functions, and moments and forces acting at the joints. A system of equations relating all such internal forces and moments is obtained. This approach was applied to simple grillages. Response results showed good agreement when compared to those provided by Finite Elements.

Ion mobility spectrometry in liquid analysis

Ion mobility spectrometry (IMS) is a straightforward, low cost method for fast and sensitive determination of organic and inorganic analytes. Originally this portable technique was applied to the determination of gas phase compounds in security and military use. Nowadays, IMS has received increasing attention in environmental and biological analysis, and in food quality determination. This thesis consists of literature review of suitable sample preparation and introduction methods for liquid matrices applicable to IMS from its early development stages to date. Thermal desorption, solid phase microextraction (SPME) and membrane extraction were examined in experimental investigations of hazardous aquatic pollutants and potential pollutants. Also the effect of different natural waters on the extraction efficiency was studied, and the utilised IMS data processing methods are discussed. Parameters such as extraction and desorption temperatures, extraction time, SPME fibre depth, SPME fibre type and salt addition were examined for the studied sample preparation and introduction methods. The observed critical parameters were extracting material and temperature. The extraction methods showed time and cost effectiveness because sampling could be performed in single step procedures and from different natural water matrices within a few minutes. Based on these experimental and theoretical studies, the most suitable method to test in the automated monitoring system is membrane extraction. In future an IMS based early warning system for monitoring water pollutants could ensure the safe supply of drinking water. IMS can also be utilised for monitoring natural waters in cases of environmental leakage or chemical accidents. When combined with sophisticated sample introduction methods, IMS possesses the potential for both on-line and on-site identification of analytes in different water matrices.