Evaluation of a novel biomaterial in the suprachoroidal space of the rabbit eye.

PURPOSE: Drug delivery to treat diseases of the posterior segment of the eye, such as choroidal neovascularization and its complications, is hampered by poor intraocular penetration and rapid elimination of the drug from the eye. The purpose of this study was to investigate the feasibility and tolerance of suprachoroidal injections of poly(ortho ester) (POE), a bioerodible and biocompatible polymer, as a biomaterial potentially useful for development of sustained drug delivery systems. METHODS: After tunnelization of the sclera, different formulations based on POE were injected (100 microL) into the suprachoroidal space of pigmented rabbits and compared with 1% sodium hyaluronate. Follow-up consisted of fundus observations, echography, fluorescein angiography, and histologic analysis over 3 weeks. RESULTS: After injection, POE spread in the suprachoroidal space at the posterior pole. It was well tolerated and progressively disappeared from the site of injection without sequelae. No bleeding or retinal detachment occurred. Echographic pictures showed that the material was present in the suprachoroidal space for 3 weeks. Angiography revealed minor pigment irregularities at the site of injection, but no retinal edema or necrosis. Histology showed that POE was well tolerated in the choroid. CONCLUSIONS: POE suprachoroidal injections, an easy, controllable, and reproducible procedure, were well tolerated in the rabbit eye. POE appears to be a promising biomaterial to deliver drugs focally to the choroid and the retina.

Influence of stationary phase and eluent properties on chromatographic separation of carbohydrates

In this thesis, the sorption and elastic properties of the cation-exchange resins were studied to explain the liquid chromatographic separation of carbohydrates. Na+, Ca2+ and La3+ form strong poly(styrene-co-divinylbenzene) (SCE) as well as Na+ and Ca2+ form weak acrylic (WCE) cation-exchange resins at different cross-link densities were treated within this work. The focus was on the effects of water-alcohol mixtures, mostly aqueous ethanol, and that of the carbohydrates. The carbohydrates examined were rhamnose, xylose, glucose, fructose, arabinose, sucrose, xylitol and sorbitol. In addition to linear chromatographic conditions, non-linear conditions more typical for industrial applications were studied. Both experimental and modeling aspectswere covered. The aqueous alcohol sorption on the cation-exchangers were experimentally determined and theoretically calculated. The sorption model includes elastic parameters, which were obtained from sorption data combined with elasticity measurements. As hydrophilic materials cation-exchangers are water selective and shrink when an organic solvent is added. At a certain deswelling degree the elastic resins go through glass transition and become as glass-like material. Theincreasing cross-link level and the valence of the counterion decrease the sorption of solvent components in the water-rich solutions. The cross-linkage or thecounterions have less effect on the water selectivity than the resin type or the used alcohol. The amount of water sorbed is higher in the WCE resin and, moreover, the WCE resin is more water selective than the corresponding SCE resin. Theincreased aliphatic part of lower alcohols tend to increase the water selectivity, i.e. the resins are more water selective in 2-propanol than in ethanol solutions. Both the sorption behavior of carbohydrates and the sorption differences between carbohydrates are considerably affected by the eluent composition and theresin characteristics. The carbohydrate sorption was experimentally examined and modeled. In all cases, sorption and moreover the separation of carbohydrates are dominated by three phenomena: partition, ligand exchange and size exclusion. The sorption of hydrophilic carbohydrates increases when alcohol is added into the eluent or when carbohydrate is able to form coordination complexes with the counterions, especially with multivalent counterions. Decreasing polarity of the eluent enhances the complex stability. Size exclusion effect is more prominent when the resin becomes tighter or carbohydrate size increases. On the other hand,the elution volumes between different sized carbohydrates decreases with the decreasing polarity of the eluent. The chromatographic separation of carbohydrateswas modeled, using rhamnose and xylose as target molecules. The thermodynamic sorption model was successfully implemented in the rate-based column model. The experimental chromatographic data were fitted by using only one adjustable parameter. In addition to the fitted data also simulated data were generated and utilized in explaining the effect of the eluent composition and of the resin characteristics on the carbohydrate separation.

Evaluation of an electronic nose to assess fruit ripeness

The main goal of our study was to see whether an artificial olfactory system can be used as a nondestructive instrument to measure fruit maturity. In order to make an objective comparison, samples measured with our electronic nose prototype were later characterized using fruit quality techniques. The cultivars chosen for the study were peaches, nectarines, apples, and pears. With peaches and nectarines, a PCA analysis on the electronic nose measurements helped to guess optimal harvest dates that were in good agreement with the ones obtained with fruit quality techniques. A good correlation between sensor signals and some fruit quality indicators was also found. With pears, the study addressed the possibility of classifying samples regarding their ripeness state after different cold storage and shelf-life periods. A PCA analysis showed good separation between samples measured after a shelf-life period of seven days and samples with four or less days. Finally, the electronic nose monitored the shelf-life ripening of apples. A good correlation between electronic nose signals and firmness, starch index, and acidity parameters was found. These results prove that electronic noses have the potential of becoming a reliable instrument to assess fruit ripeness.

Evaluation of Xylella fastidiosa genetic diversity by fAFPL markers

The first phytopathogenic bacterium with its DNA entirely sequenced is being detected and isolated from different host plants in several geographic regions. Although it causes diseases in cultures of economic importance, such as citrus, coffee, and grapevine little is known about the genetic relationships among different strains. Actually, all strains are grouped as a single species, Xylella fastidiosa, despite colonizing different hosts, developing symptoms, and different physiological and microbiological observed conditions. The existence of genetic diversity among X. fastidiosa strains was detected by different methodological techniques, since cultural to molecular methods. However, little is know about the phylogenetic relationships developed by Brazilian strains obtained from coffee and citrus plants. In order to evaluate it, fAFLP markers were used to verify genetic diversity and phylogenetic relationships developed by Brazilian and strange strains. fAFLP is an efficient technique, with high reproducibility that is currently used for bacterial typing and classification. The obtained results showed that Brazilian strains present genetic diversity and that the strains from this study were grouped distinctly according host and geographical origin like citrus-coffee, temecula-grapevine-mulberry and plum-elm.

Evaluation of substrates on the emergence of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) Seedlings

Choosing a substrate is the determinant factor for the seedling producer; thus, the aim of this study was to evaluate the effect of different types of substrates on the emergence of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) seedlings. The experiment was carried out in a greenhouse and the experimental design was in randomized blocks, with three treatments and five replicates of 72 seeds per plot. The treatments consisted of the following substrates: coconut fiber, vermiculite and Plantmax® Citrus. The number of emerged seedlings was weekly counted for 105 days. Data regarding seedling height were obtained, and the emergence velocity index and mean time, besides total emergence percentage and that over time were calculated. Results from total mean emergence percentage, seedling height, emergence velocity index (EVI), and mean emergence time (MET) were subjected to analysis of variance and means were compared by the Tukey's test at 5% significance. The curves concerning the emergence percentage over time were fit by the logistic growth equation for each treatment and the means of each parameter (A, B, C) were compared by the Duncan's test at 5% significance. The substrates vermiculite led to the highest values of emergence percentage differing from the PlantMax® Citrus, but not of the coconut fiber, however the vermiculite promoted seedling height in a shorter time; therefore, this substrate is recommended for the initial development of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) seedlings.

A nursing interface terminology: evaluation of face validity

A range of different language systems for nursing diagnosis, interventions and outcomes are currently available. Nursing terminologies are intended to support nursing practice but they have to be evaluated. This study aims to assess the results of an expert survey to establish the face validity of a nursing interface terminology. The study applied a descriptive design with a cross-sectional survey strategy using a written questionnaire administered to expert nurses working in hospitals. Sample size was estimated at 35 participants. The questionnaire included topics related to validity and reliability criteria for nursing controlled vocabularies described in the literature. Mean global score and criteria scoring at least 7 were considered main outcome measures. The analysis included descriptive statistics with a confidence level of 95%. The mean global score was 8.1. The mean score for the validity criteria was 8.4 and 7.8 for reliability and applicability criteria. Two of the criteria for reliability and applicability evaluation did not achieve minimum scores. According to the experts" responses, this terminology meets face validity, but that improvements are required in some criteria and further research is needed to completely demonstrate its metric properties.

Evaluation of winter temperatures on apple budbreak using grafted twigs

Temperature is the main climate factor related to induction, maintenance and dormancy release in apple (Malus domestica Borkh.). The inadequate chilling exposure in apples causes budbreak problems, resulting in decrease in yield potential. Thus, the knowledge of physiological principles and environmental factors determining the dormancy phenomenon, especially winter temperature effects, it is necessary for the efficient selection of cultivars in a productive region. In addition, it is indispensable to adapt the orchard management aiming to decrease the problems caused by lack chilling during winter. The objective of this study was to evaluate the influence of different thermal conditions during the dormancy period on budbreak of apple cultivars. One-year-old twigs of 'Castel Gala' and 'Royal Gala' cultivars, grafted on M7 rootstock, were submitted to temperatures of 5, 10 and 15ºC for different exposure periods (168; 336; 672; 1,008 and 1,344 hours). After treatments execution, the plants were kept in a greenhouse at 25ºC. Budbreak was quantified when accumulated 3,444; 6,888; 10,332; 13,776; 17,220 and 20,664 GDHºC after temperature treatments. The cultivars responded differently to temperature effect during the winter period. The temperature of 15ºC during winter shows a greater effectiveness on 'Castel Gala' apple budbreak while in the 'Royal Gala' apples the temperatures of 5 and 10ºC show better performance. 'Castel Gala' cultivar (low chilling requirement) may supply its physiological necessities, may be capable to budburst, even when subjected to higher temperatures in relation to 'Royal Gala' apples (high chilling requirement).

An Evaluation of the Paper Profile - a comparative assessment of the applicability of an emerging environmental communication tool in pulp and paper industry

Diplomityön tavoitteena oli arvioida uutta sellu-, paperi- ja kartonkiteollisuuden Paper Profile -ympäristötuoteselostetta. Paper Profilen tarkoituksena on tarjota paperiteollisuuden asiakkaille ja muille kiinnostuneilla sidosryhmille yhtenäistä ympäristöinformaatiota koskien paperituotteiden koostumusta ja tuotteiden tärkeimpiä ympäristöparametrejä. Työn tärkein tavoite oli arvioida kriittisesti Paper Profilea ja verrata konseptia ISO:n, Kansainvälisen stardardisoimisliiton tuoteselosteeseen sekä löytää konseptien yhtäläisyydet ja erot. Tärkein tehtävä oli tunnistaa ne avaintekijät, joiden avulla Paper Profile -tuoteselostetta voitaisiin pitää yhtenevänä ISO/TR 14025 teknisen raportin kanssa. Lisäksi Paper Profile -tuoteselosteen mahdolliset kehittämistarpeet arvioitiin ISO-tuoteselosteen näkökulmasta. Työn toinen tavoite oli kerätä ja analysoida uuteen tuoteselosteeseen liittyvä asiakaspalaute ja verrata sitä Stora Enson tehtaiden ympäristöpäälliköiden antamiin Paper Profilea koskeviin kommentteihin. Työn tuloksena huomattiin, että Paper Profile -konsepti sellaisenaan ei ole kovin informatiivinen vaan tuoteseloste jättää monia ympäristökysymyksiä avoimeksi. Siitä huolimatta Paper Profile tarjoaa riittävän taustan eri paperituotteiden ympäristökuormitusten keskinäiselle vertailuille. Konseptin viestinnällinen näkökulma paranisi huomattavasti, jos tuoteselosteeseen lisättäisiin kolmannen osapuolen verifiointi. Lisäksi referenssiarvot kertoisivat asiakkaille paremmin esitettyjen parametrien taustoista. Stora Enson tehtaiden ympäristöpäälliköiden, samoin kuin asiakkaidenkin mielestä Paper Profile on yleisesti ottaen potentiaalinen ympäristöviestinnän työkalu, mutta silti konseptiin ehdotettiin joitakin pieniä muutoksia. Avoinna olevat metsäsertifiointikysymykset ja niiden puutteellinen tiedottaminen tuoteselosteessa puhututtivat sekä tehtaiden henkilöstöä että yritysasiakkaita.

BS-SEM evaluation of the tisular interactions between cortical bone and calcium-phosphate covered titanium implants

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes

BS-SEM evaluation of the tisular interactions between cortical bone and calcium-phosphate covered titanium implants

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes

BS-SEM evaluation of the tisular interactions between cortical bone and calcium-phosphate covered titanium implants

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes

Evaluation of steroidomics by liquid chromatography hyphenated to mass spectrometry as a powerful analytical strategy for measuring human steroid perturbations.

This review presents the evolution of steroid analytical techniques, including gas chromatography coupled to mass spectrometry (GC-MS), immunoassay (IA) and targeted liquid chromatography coupled to mass spectrometry (LC-MS), and it evaluates the potential of extended steroid profiles by a metabolomics-based approach, namely steroidomics. Steroids regulate essential biological functions including growth and reproduction, and perturbations of the steroid homeostasis can generate serious physiological issues; therefore, specific and sensitive methods have been developed to measure steroid concentrations. GC-MS measuring several steroids simultaneously was considered the first historical standard method for analysis. Steroids were then quantified by immunoassay, allowing a higher throughput; however, major drawbacks included the measurement of a single compound instead of a panel and cross-reactivity reactions. Targeted LC-MS methods with selected reaction monitoring (SRM) were then introduced for quantifying a small steroid subset without the problems of cross-reactivity. The next step was the integration of metabolomic approaches in the context of steroid analyses. As metabolomics tends to identify and quantify all the metabolites (i.e., the metabolome) in a specific system, appropriate strategies were proposed for discovering new biomarkers. Steroidomics, defined as the untargeted analysis of the steroid content in a sample, was implemented in several fields, including doping analysis, clinical studies, in vivo or in vitro toxicology assays, and more. This review discusses the current analytical methods for assessing steroid changes and compares them to steroidomics. Steroids, their pathways, their implications in diseases and the biological matrices in which they are analysed will first be described. Then, the different analytical strategies will be presented with a focus on their ability to obtain relevant information on the steroid pattern. The future technical requirements for improving steroid analysis will also be presented.

Evaluation of Teacher Education Programs by Students and Graduates

The purpose of this research was to do a repeated cross-sectional research on class teachers who study in the 4th year and also graduated at the Faculty of Education, University of Turku between the years of 2000 through 2004. Specifically, seven research questions were addressed to target the main purpose of the study: How do class teacher education masters’ degree senior students and graduates rate “importance; effectiveness; and quality” of training they have received at the Faculty of Education? Are there significant differences between overall ratings of importance; effectiveness and quality of training by year of graduation, sex, and age (for graduates) and sex and age (for senior students)? Is there significant relationship between respondents’ overall ratings of importance; effectiveness and their overall ratings of the quality of training and preparation they have received? Are there significant differences between graduates and senior students about importance, effectiveness, and quality of teacher education programs? And what do teachers’ [Graduates] believe about how increasing work experience has changed their opinions of their preservice training? Moreover the following concepts related to the instructional activities were studied: critical thinking skills, communication skills, attention to ethics, curriculum and instruction (planning), role of teacher and teaching knowledge, assessment skills, attention to continuous professional development, subject matters knowledge, knowledge of learning environment, and using educational technology. Researcher also tried to find influence of some moderator variables e.g. year of graduation, sex, and age on the dependent and independent variables. This study consisted of two questionnaires (a structured likert-scale and an open ended questionnaire). The population in study 1 was all senior students and 2000-2004 class teacher education masters’ degree from the departments of Teacher Education Faculty of Education at University of Turku. Of the 1020 students and graduates the researcher was able to find current addresses of 675 of the subjects and of the 675 graduates contacted, 439 or 66.2 percent responded to the survey. The population in study 2 was all class teachers who graduated from Turku University and now work in the few basic schools (59 Schools) in South- West Finland. 257 teachers answered to the open ended web-based questions. SPSS was used to produce standard deviations; Analysis of Variance; Pearson Product Moment Correlation (r); T-test; ANOVA, Bonferroni post-hoc test; and Polynomial Contrast tests meant to analyze linear trend. An alpha level of .05 was used to determine statistical significance. The results of the study showed that: A majority of the respondents (graduates and students) rated the overall importance, effectiveness and quality of the teacher education programs as important, effective and good. Generally speaking there were only a few significant differences between the cohorts and groups related to the background variables (gender, age). The different cohorts were rating the quality of the programs very similarly but some differences between the cohorts were found in the importance and effectiveness ratings. Graduates of 2001 and 2002 rated the importance of the program significantly higher than 2000 graduates. The effectiveness of the programs was rated significantly higher by 2001 and 2003 graduates than other groups. In spite of these individual differences between cohorts there were no linear trends among the year cohorts in any measure. In respondents’ ratings of the effectiveness of teacher education programs there was significant difference between males and females; females rated it higher than males. There were no significant differences between males’ and females’ ratings of the importance and quality of programs. In the ratings there was only one difference between age groups. Older graduates (35 years or older) rated the importance of the teacher training significantly higher that 25-35 years old graduates. In graduates’ ratings there were positive but relatively low correlations between all variables related to importance, effectiveness and quality of Teacher Education Programs. Generally speaking students’ ratings about importance, effectiveness and quality of teacher education program were very positive. There was only one significant difference related to the background variables. Females rated higher the effectiveness of the program. The comparison of students’ and graduates’ perception about importance, effectiveness, and quality of teacher education programs showed that there were no significant differences between graduates and students in the overall ratings. However there were differences in some individual variables. Students rated higher in importance of “Continuous Professional Development”, effectiveness of “Critical Thinking Skills” and “Using Educational Technology” and quality of “Advice received from the advisor”. Graduates rated higher in importance of “Knowledge of Learning Environment” and effectiveness of “Continuous Professional Development”. According to the qualitative data of study 2 some graduates expressed that their perceptions have not changed about the importance, effectiveness, and quality of training that they received during their study time. They pointed out that teacher education programs have provided them the basic theoretical/formal knowledge and some training of practical routines. However, a majority of the teachers seems to have somewhat critical opinions about the teacher education. These teachers were not satisfied with teacher education programs because they argued that the programs failed to meet their practical demands in different everyday situations of the classroom e.g. in coping with students’ learning difficulties, multiprofessional communication with parents and other professional groups (psychologists and social workers), and classroom management problems. Participants also emphasized more practice oriented knowledge of subject matter, evaluation methods and teachers’ rights and responsibilities. Therefore, they (54.1% of participants) suggested that teacher education departments should provide more practice-based courses and programs as well as closer collaboration between regular schools and teacher education departments in order to fill gap between theory and practice.

Evaluation of molecular, cellular and behavioral consequences of serotonin 1A receptor deletion

In 1998, three different research groups simultaneously reported increased anxiety-related behavior in tests of conflict in their serotonin 1a (5-HT1a) receptor knockout (KO) line with male mice being more severely affected by 5-HT1a receptor deletion than female KO. Similarly, in the hippocampus, we observed increased dendritic complexity in the stratum radiatum of CA1 pyramidal neurons in male but not in female 5-HT1a receptor KO mice. These observations prompted us to investigate gender- dependent differences of 5-HT1a receptor deletion in hippocampal-related behavioral tasks. Testing our mice in anxiety-related paradigms, we reproduced the original studies showing increased anxiety- related behavior in male 5-HT1a receptor KO mice when compared to male WT mice, but no difference between female 5-HT1a receptor KO and WT mice. Similarly, male 5-HT1a receptor KO mice were impaired in association of aversive stimuli fear conditioning paradigms. We argue that increased dendritic complexity and increased synaptic strength of CA3-CA1 synapses in the stratum radiatum impaired proper signal propagation attributed to overactivation of CA1 pyramidal neurons leading to impaired fear memory of male 5-HT1a receptor KO mice. Similar mechanisms in the ventral hippocampus are likely to have contributed to gender-dependent differences in anxiety-related behavior in our and the original studies from 1998. In this study, we started to shed light on the 5-HT1a receptor downstream signaling pathways involved in dendritogenesis of pyramidal neurons during early postnatal development. We could show that NR2B-containing NMDA receptor during development acts downstream of 5-HT1a receptor and is responsible for increased amount of branching in male 5-HT1a receptor KO mice. Conversely, protein and NR2B mRNA expression was increased in 5-HT1a receptor KO mice at P15. Although the exact signaling cascade of 5-HT1a receptor regulating NR2B-containing NMDA receptor has not been determined, CaMKII is a potential downstream effector to influence transportation and removal of NR2B-containing NMDA receptors to and from the synapse. In contrast, Erk1/2 likely acts downstream of NR2B-containing NMDA receptors and was shown to be sufficient to regulate dendritic branching. Moreover, increased NR2B-containing NMDA receptor mediated cell death via excitotoxicity during development and is likely to be involved in reduced survival of adult born neurons in the hippocampus of 5-HT1a receptor KO male. The convergence of 5-HT1a receptor signaling onto NR2B-containing NMDA receptor signaling enables estrogen to interfere with its downstream pathway via G-protein coupled estrogen receptor 1 activation resulting in normalization of branching and behavior in female 5-HT1a receptor mice. In conclusion, our data strongly suggests a hormone- regulated mechanism that by converging on NR2B-containing NMDA receptor signaling is able to normalize morphology of pyramidal neurons and behavior of female 5-HT1a receptor KO mice. Our findings provide a possible explanation for gender-dependent differences in the occurrence of mental disorders with 5-HT1a receptor abnormalities as a strong predisposing factor. -- En 1998, trois équipes de recherche ont décrit un comportement de type anxieux dans des tests de conflit pour leur souris transgéniques avec une délétion du gène pour le récepteur 5-HT1a de la sérotonine. De plus, les trois groupes rapportent un phénotype plus sévère pour le comportement anxieux chez les souris transgéniques mâles que femelles. Dans l'hippocampe, la région avec la densité de récepteur 5-HT1a la plus élevée dans le télencéphale, nous avons observé dans le stratum radiatum une complexité accrue des arborisations dendritiques des neurones pyramidaux du secteur CA1 chez les souris transgénique mâles mais pas chez les femelles. Cette observation nous a encouragés à initier cette étude sur les différences en fonction du genre utilisant les tests comportementaux en rapport avec les fonctions de l'hippocampe chez les souris déficientes pour le récepteur 5-HT1a.Testant nos souris avec des paradigmes associés à l'anxiété, nous avons reproduit les données originales montrant que les souris transgéniques mâles ont un phénotype plus sévère que les souris mâles sauvages, mais qu'aucune différence n'est observée entre les femelles sauvages et transgéniques. De même, les souris mâles déficientes pour le récepteur 5-HT1a sont handicapées dans les tests de conditionnement au stress avec des stimuli aversifs. Nous faisons l'hypothèse que l'augmentation de la complexité de l'arborisation dendritique et l'augmentation de la force du signal synaptique entres les régions CA3 et CA1 de l'hippocampe dans le stratum radiatum perturbe la propagation du signal nerveux qui conduit à l'hyperactivation des neurones du secteur CA1. Ceci conduit à une mémoire de stress altérée chez les souris mâles déficientes pour le récepteur 5-HT1a. Un mécanisme similaire dans l'hippocampe ventral contribue probablement aux différences en fonction du genre dans les tests pour le comportement de type anxieux qui ont été rapportés dans les études originales de 1998. Les mesures de protéine et de mRNA ont mis en évidence une augmentation de l'expression du récepteur NMDA contenant la sous- unité NR2B dans les souris déficientes pour le récepteur 5-HT1a à P15. Dans les cultures organotypiques d'hippocampe, nous avons commencé à disséquer les messagers secondaires à l'activation du récepteur 5-HT1a qui sont impliqués dans la régulation de la croissance dendritique des neurones pyramidaux pendant la période postnatale précoce. Nous avons démontré que les récepteurs NR2B sont en aval de l'activation du récepteur 5-HT1a et qu'ils sont impliqués dans l'accroissement du nombre de dendrites chez la souris mâle déficiente pour le récepteur 5-HT1a. Bien que la cascade de signalisation du récepteur 5-HT1a pour réguler les récepteurs NMDA contenant le NR2B ne soit pas établie, CaMKII est identifié comme un effecteur potentiel pour altérer le transport du récepteur NMDA à la synapse. D'autre part, Erk1/2 est probablement un messager en aval du NR2B du récepteur NMDA, et a été documenté comme suffisant pour réguler l'arborisation dendritique. L'augmentation de NR2B à la synapse des souris déficientes pour le récepteur 5-HT1a peut conduire à une augmentation de l'excitotoxicité dans les cellules. Nous avons observé une augmentation chez la souris déficiente pour le récepteur 5-HT1a de la mort cellulaire dans des tranches d'hippocampe stimulées, ce qui peut être en relation avec la réduction de la survie des neurones générés dans l'hippocampe de la souris mâle transgénique adulte par rapport à la souris mâle sauvage. De plus, la convergence de la signalisation du récepteur 5-HT1a sur la signalisation de la sous-unité NR2B du récepteur NMDA permet à l'oestrogène d'interférer avec sa voie de signalisation du récepteur de l'oestrogène couplé à une protéine G (GPER-1), ceci permettant à l'oestrogène de réduire la taille de l'arborisation des neurones pyramidaux de CA1 chez la femelle de la souris déficiente pour le récepteur 5-HT1a. En conclusion, nos observations suggèrent fortement qu'un mécanisme hormonal convergeant sur la voie de signalisation de la sous-unité NR2B du récepteur NMDA permet la normalisation de l'exubérance des dendrites des neurones CA1 de l'hippocampe et du comportement des souris femelles déficientes pour le récepteur 5-HT1a. Ceci donne une explication possible pour la différence en fonction du genre dans l'apparition de troubles mentaux avec les variations du récepteur 5-HT1a comme facteur de prédisposition important.

Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.