Osteoporosis is a disease of the bone metabolism which is characterised with a decrease of bone substance. The cause of this disease is the imbalance between the creation of a new bone substance by osteoblasts and the resorption of a bone tissue by osteoclasts, in favour of the bone resorption. The risk group of the development of this disease are women after menopause, who naturally register a decline of the estrogen hormone. Estrogen operates as an inhibitor of proosteoclastic factors such as receptor activator of NFκB ligand (RANKL), interleukin (IL)-1, IL-6 or TNF-α. The imbalance of the bone metabolism can also be caused by a disbalance in the production of Prostaglandin E2 (PGE2) and 1α,25-dihydroxyvitamin D3. They are strong mediators which can both stimulate and inhibit an osteoclastogenesis in vitro in concordance with the conditions of the culture/co-culture. This thesis focuses on the examination of an influence of those mediators (PGE2 in the concentration of 10-6 M and 10-8 M; 1α,25-dihydroxyvitamin D3 in the concentration of 10-8 M and 10-9 M) on the osteoclastogenesis from the rat PBMC at the presence of osteoblasts, with or without the combination of proosteoclastic factors macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclastogenesis was stimulated if PGE2 and...

Plants are a source of a wide range of secondary substances, which due to their effects find use in many areas of focus. By a method called elicitation, we can achieve their higher and thus more efficient production. This diploma thesis aimed to determine whether the abiotic elicitor 2-(4-chlorophenyl)-N-(5-chloropyridin-2-yl)acetamide can positively affect the production of the flavonoid hyperoside in callus and suspension culture of Hypericum perforatum L. The elicitor was added to the in vitro cultures in three concentrations: C1 = 3,571.10-3 mol/l; C2 = 3,571.10-4 mol/l and C3 = 3,571.10-5 mol/l. A sample was taken at regular intervals after 6, 24, 48, 72 and 168 hours of elicitor treatment. Control samples were taken after 24 and 168 hours. The content of hyperoside produced was subsequently determined using High Performance Liquid Chromatography. Simultaneously, the amount of hyperoside released into the nutrient media of both plant cultures was also monitored. Maximum hyperoside production was recorded in suspension culture after 6 (17,7 µg/g DW) and 48 hours (3,69 µg/g DW) of elicitor treatment with the lowest concentration of C3 (3,571.10-5 mol/l). The content of hyperoside in the first case was 1770 % higher compared to the control sample. There was a significant release of hyperoside...

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Memoria para optar al Título Profesional de Médico Veterinario / En caninos, el desarrollo de las biotecnologías reproductivas ha sido inferior en comparación al realizado en otras especies, básicamente debido a la baja eficiencia en la maduración in vitro de los ovocitos. La adecuada maduración comprende tanto al núcleo como al citoplasma, a nivel citoplasmático involucra la redistribución y desarrollo de organelos, entre estos, la organización y la actividad metabólica de las mitocondrias. Sin embargo, no hay mayores antecedentes respecto al desarrollo citoplasmático in vitro en los caninos, por lo que en la presente memoria se estudió la actividad y distribución mitocondrial en ovocitos de perras en estado inmaduro, madurados in vitro por 72 y 96h y madurados in vivo. Se analizó un total de 766 ovocitos obtenidos de perras sanas sometidas a ovariohisterectomía. Del total de estos ovocitos estudiados, 215 fueron utilizados como inmaduros, 254 fueron madurados durante 72 horas y los otros 297 durante 96 horas. Adicionalmente se analizó una cantidad total de 15 ovocitos madurados in vivo, obtenidos por lavados del oviducto a perras en estro, ovariohisterectomizadas 72 h después de la ovulación, la que se determinó mediante análisis de progesterona sérica. Tanto los ovocitos inmaduros, madurados in vitro y madurados in vivo fueron procesados de forma separada para evaluar la distribución y actividad mitocondrial, usando la sonda de fluorescencia MitoTracker Red CMXRos®. Las observaciones se realizaron en un microscopio invertido con epifluorescencia. La actividad mitocondrial se evaluó de acuerdo a la fluorescencia emitida en alta, media y baja para cada uno de los estados de maduración de los ovocitos y se analizó mediante chi-cuadrado. Se estableció dependencia entre las frecuencias de ovocitos con distintas intensidades de fluorescencia (alta, media, baja) al comparar las muestras provenientes de ovocitos inmaduros, madurados in vitro por 72 y 96 h, y madurados in vivo (chi cuadrado = 30,59; p< 0,01), indicando que la actividad mitocondrial se encuentra directamente asociada al estado de maduración del ovocito de perra. En la actividad mitocondrial todos los ovocitos madurados in vivo presentaron fluorescencia de tipo media, la que predominó en ovocitos de perra inmaduros y madurados in vitro; sin embargo, se observó un aumento en el porcentaje de ovocitos con 3 esta emisión de fluorescencia durante la maduración in vitro que indicaría una actividad mitocondrial progresiva durante el cultivo, asociada al tiempo de maduración. La distribución mitocondrial se evaluó mediante análisis descriptivo, determinándose cuatro patrones: Homogéneo liso completo (A), Homogéneo granuloso completo (B), Heterogéneo granuloso periférico y central (C), y Heterogéneo granuloso central (D). En ovocitos inmaduros se encontraron dos patrones de distribución mitocondrial A (72,1%) y B (27,9%); en aquellos madurados in vitro por 72 h se presentaron los cuatro patrones, predominando el B (63,8%), seguido por C (20,5%), el A (9,8%) y finalmente el D (5,9%). en los ovocitos madurados por 96 h se observaron los patrones, B (63,6%), seguido por C (35,7%) y finalmente D (0,7%). En el caso de los ovocitos madurados in vivo se describió sólo un patrón de distribución mitocondrial que correspondió a B. Con estos antecedentes se indica que en ovocitos inmaduros de perra predomina una distribución uniforme de las mitocondrias en su citoplasma. Además la presentación de tres patrones diferentes en los ovocitos madurados in vitro a aquel descrito en ovocitos madurados in vivo podría indicar que durante la maduración in vitro se producen movimientos de las mitocondrias en el citoplasma ovular. También, las distribuciones de las mitocondrias, variaron a través de la maduración in vitro, asemejándose a aquellos madurados in vivo, sin embargo, hubo diferencias entre ambos tipos de maduración lo que podría estar asociado a las condiciones de cultivo. / Proyecto FONDECYT 1080618

In einer vorangegangenen In-vitro-Untersuchung wurden die pallatinalen Wurzeln menschlicher Molaren im Scherversuch (10mm/Min) gebrochen. Zahnfragmente wurden mit Hilfe verschiedener Dentinhaftvermittler (OptiBond FL, Syntac, Adhese, Prompt L-Pop und OptiBond FL in Kombination mit Tetrac Flow) adhäsiv wiederbefestigt. Die so rekonstruierte Zahneinheit wurde erneut an der ursprünglichen Bruchfläche gebrochen und die Bruchfestigkeit ermittelt. In der vorliegenden In-vitro-Untersuchung wurden die Bruchflächen der erneut gebrochenen Zähne digital abfotografiert und lichtmikroskopisch untersucht, um den Frakturmodus zu ermitteln. Die Frakturmodi (Adhäsivfraktur, Kohäsivfraktur im Dentin oder Befestigungsmaterial) wurden absolut und in Relation zur gesamten Bruchfläche ermittelt. Hierzu wurde ein spezielles CAD-Programm verwendet. Mittels statistischer Tests wurden die Adhäsivsysteme auf signifikante Unterschiede hinsichtlich der aufgetretenen Frakturmodi untersucht. Ergebnisse: Keine signifikanten Unterschiede. Kohäsivfrakturen im Befestigungsmaterial traten unabhängig vom Dentinhaftvermittler am häufigsten auf. Vor allem konnte dieser Bruchmodus bei Verwendung von OptiBond FL beobachtet werden. Adhäsivfrakturen traten am zahlreichsten bei Gebrauch von Prompt L-Pop und Kohäsivfrakturen im Dentin bei Verwendung von Syntac auf. / In a preceding in-vitro-investigation roots of human molars were broken in shear-test (10mm/min). Teeth were restored by fragment fixation with different adhesive systems (OptiBond FL, Syntac, Adhese, Prompt L-Pop and OptiBond FL in combination with Tetrac Flow) and shear test was repeated to investigate fracture-toughness. In this in-vitro investigation pictures of the surfaces of the broken roots were taken and they were analysed using light-microscope. Failure-modes (adhesive-fracture, cohesive-fracture in dentine or restaurative material) were calculated absolute and in relation to the whole surface by a special CAD-software. Statistical tests were done to find out significant difference in failure-mode between the tested adhesive systems. Results: No significant differences. In every adhesive system cohesive fracture in restaurative material was the most frequent fracture-mode. Most of them could be observed if OptiBond FL was used. Adhesive fractures especially appeared in the group of Prompt L-Pop, and cohesive fractures in dentine by using syntac.

In Deutschland und weltweit, werden mehr und mehr, neben einem makellosen Körper und Gesicht, auch makellose Zähne als Schönheits- und Statussymbol betont. Sogar können die kleinste Verfärbungen oder Füllungen Grund zur Besorgnis für Erwachsene, Kinder und Eltern sein. Als Therapie in der Zahnheilkunde ist verbreitet, diese Verfärbungen bzw. Flecken mit Kompositfüllungen, Veneers, Kronen oder mit abrasiven Schleifkörpern invasiv beseitigen zu können. Weniger angewendet, doch erste Wahl der Therapie sollte die minimal invasive Mikroabrasionsbehandlung sein. Dabei werden mit abrasiven und säurehaltigen Pasten die innerhalb der Oberfläche liegenden Verfärbungen beseitigt. Über die Größe des Schmelzabtrages in der zahnärztlichen Literatur findet man nur geringe metrische Angaben. Deshalb ist Ziel dieser vorliegenden Studie die Untersuchung des Schmelzsubstanzabtrages durch Mikroabrasion mit der Mikroabrasionspaste Opalustre® in den Bearbeitungszeiten von 5, 50 und 100 Sekunden, mit gleich bleibendem Anpressdruck und Umdrehungszahl und so herauszufinden, ob bei konstantem Druck und Umdrehungszahl die Größe des Abriebs mit der Behandlungszeit korreliert. Humane extrahierte Zähne dienten dabei als In-vitro-Testsystem. Die Analyse des Schmelzabtrages erfolgte an Dünnschnitten der behandelten humanen Zähne unter einem Lichtmikroskop bei 50 facher Vergrößerung. Auf die Labialflächen von 21 extrahierten und in 0,1 %-iger Thymollösung gelegten Zähnen wurde eine eingefärbte Bondingschicht mittig als Referenzfläche aufgetragen. Die in drei Versuchsgruppen aufgeteilten Zähne wurden dann mit der Mikroabrasionspaste Opalustre® für 5, 50 und 100 Sekunden bei einem Druck von etwa 200 g, bei etwa 135 U/min (Kavo-Reduzierwinkelstück, Doppelring grün 7,4:1 bei 1000 U/min) mit der Paste und dem dazugehörigen Polierkelch (Opal Cups-Bristle™) mikroabradiert. Nach Einbetten der Zahnkronen in Kunststoffblöcke wurden sie in ca. 0,7 mm (± 0,1mm) dicke Scheiben (Proben) geschnitten und unter dem Lichtmikroskop quantitativ nach dem Schmelzabtrag untersucht. Der Mittelwert des Substanzabtrages bei 5 Sekunden Bearbeitungszeit lag bei 14,80 µm, bei 50 Sekunden bei 20,86 µm und bei 100 Sekunden bei 23,74 µm. Die Ergebnisse sind signifikant. Damit wurde gezeigt, daß eine Anwendungszeit von 50 oder 100 Sekunden nicht effektiver ist als 5 Sekunden. Ein häufiger 5 Sekunden Wechsel mit wiederholt neuem Auftragen frischer Mikroabrasionspaste, entgegen der Herstellerangaben, zeigt einen effizienteren Abtrag als eine Zeitverlängerung. Für Fluorose bedingte Verfärbungen 2. Grades könnte somit in ca. 30 Sekunden mit Opalustre® (bei einer Umdrehungszahl von 135 rpm und einem Druck von 200 g) ein genügend tiefer Abtrag des betroffenen Schmelzareals (ca. 100 µm) mit dem gelieferten Kelch (OpalCups-Bristle™) stattfinden, wenn alle 5 Sekunden die Paste neu aufgetragen wird. / In Germany and throughout the world dental esthetics is as important to perception of beauty as an immaculate body and face. Even the smallest discoloration or filling can be cause for concern for adults, children and parents. When dental therapy is needed common methods to treat these discolorations and/or marks include composite-fillings, veneers, or use of abrasive grinding wheels. Less applied, but first choice of the therapy should be the minimum invasive micro abrasion treatment. With abrasion and acid pastes the discolorations lying within the surface are eliminated. The present study investigates the fusion substance erosion by micro abrasion with the micro abrasion paste Opalustre®. Varying operation times of 5, 50 and 100 seconds to determine how contact pressure and number of revolutions effects the size of the abrasion and how this correlates with the treatment time. Human extracted teeth served as the in-vitro-test system. The analysis of the fusion erosion was performed on thin sections of the human teeth treated under an optical microscope with 50 times magnification. On the labial surface of 21 extracted and in 0,1% Thymol teeth were laid on a dyed bonding layer centrically as reference surface. The teeth were divided into three experimental groups and were then microabraded with the micro abrasion paste Opalustre® for 5, 50 and 100 seconds with a pressure by approximately 200 g, with approximately 135 rpm (Kavo gear reduction handpiece double ring green 7,4:1 with 1000 rpm) with the paste and the pertinent polishing cup (Opal Cups Bristle™). After embedding the tooth crowns into plastic blocks they were cut in approx. 0.7 mm (± 0,1mm) thick disks (samples) and examined under the optical microscope quantitatively after the fusion erosion. The average value of the substance erosion at 5 seconds of operating time was with 14,80 µm, at 50 seconds with 20,86 µm and at 100 seconds with 23,74 µm. The results are significant. For Fluorose would know conditioned discolorations of 2nd degree thus in approx. 30 seconds with Opalustre® (at a number of revolutions of the concerned of the fusion area by 135 rpm and a pressure of 200 g) a sufficient a deep erosion (approx. 100 µm) with the supplied cup (Opal Cups Bristle™) to take place, if every 5 seconds the paste is again laid on.

Chez Arabidopsis thaliana, deux gènes codent des enzymes de branchement (BE) BE2.2 et BE2.1 responsables de la formation des liaisons α-1,6 de l’amidon transitoire synthétisé dans la feuille de la plante. Ces enzymes, appartenant à la famille GH13_8 de la classification CAZy, agissent en clivant une liaison α-1,4 d’un glucane puis en transférant la chaîne clivée en position α-1,6 selon un mécanisme d’action qui peut être intra ou intermoléculaire. Dans ce travail, une caractérisation enzymatique et structurale des BEs d’A. thaliana (classées de type II d’après leur séquence en acides aminés) a été réalisée et les résultats comparés à ceux de la BE d’E. coli (GlgB, enzyme de type I).L'état oligomérique, la forme en solution et l’organisation structurale des BEs ont été évalués par une approche SAXS. Par des analyses spectrophotométriques, le pH, la température, mais aussi le KM pour l’amylose et l’amylopectine ont été déterminés. Une analyse sur gel de polyacrylamide, en conditions natives, a permis d’évaluer le comportement électrophorétique des BEs en présence ou en absence de ces substrats et d’en déterminer leur constante d’affinité (Ks). Notre étude révèle que les BEs d’ A. thaliana ont plus d'affinité pour l’amylopectine que pour l’amylose contrairement à GlgB. En présence d’un substrat branché, des changements d’oligomérie et/ou de la conformation des BEs d’A. thaliana ont été observés. Finalement, des analyses en chromatographie échangeuse d'anions ont permis de déterminer la taille minimale du substrat nécessaire à l’activité des BEs et la taille des chaînes transférées. Les résultats obtenus pointent vers un mécanisme d’action intramoléculaire de BE2.2. / BE2.2 and BE2.1 are the two genetically independent branching enzymes (BE) isoforms involved in transitory starch synthesis in A. thaliana and belong to family GH13_8 (according to CAZy database). Both are classified as type II BE due to their amino acid sequence. In Arabidopsis leaves, they are the only enzymes that catalyze the formation of α-1,6 branch points by cleaving α-1,4 linkages and transferring the newly formed reducing end in α-1,6 position through an intra or intermolecular mechanism. In this work, we report in vitro enzymatic characterization and structural analysis of A. thaliana BEs, these results were compared to E. coli BE enzymatic analysis (GlgB, type I enzyme).Structural analysis using SAXS approach was used to evaluate A. thaliana BEs oligomeric state, shape in solution and to determine BE organization. In vitro enzymatic analyses were performed using spectrophotometry assays to establish their catalytic parameters such as pH, temperature and also KM for amylose and amylopectin. Native PAGE analyses were also used to assess BEs behaviour in the presence or absence of substrates and to determine their affinity constant (Ks) for amylopectin and amylose. Enzymatic characterization reveals that both A. thaliana BEs have more affinity for amylopectin than for amylose, contrary to GlgB. Moreover, interaction of A. thaliana BEs with branched substrates induces protein oligomerization and/or conformational changes. Finally, determination of the minimal length of their substrate and characterization of reaction products were performed using anions exchange chromatography analyses.Taken together, our data point to an intramolecular mechanism of action of BE2.2.

Osteoarthritis (OA) is a chronic, degenerative condition of articular joints. The prevalence of OA is high in many mammalian populations, though our understanding of the disease is limited, with the initiating factors and the early phenotype of the disease being poorly characterised. Clinically, the early-stage of OA is rarely identified, precluding the identification and treatment of affected individuals. Consequently, in vitro models of OA typically reflect the later stages of the disease, and are rarely validated against the naturally-occurring disease. This project utilised tissue from a naturally-occurring canine disease (medial coronoid process disease) to characterise the transcriptome of early-stage OA, and inform different in vitro models, to try and refine the model conditions. Medial coronoid processes from affected dogs were removed and graded histologically, both manually and through the development of a semi-automated assessment. Early-stage OA was characterised by a decrease in the chondrocyte density, an increase in the thickness of the articular cartilage and a loss of proteoglycan. No histological changes in bone morphology were noted in early-stage OA. A transcriptomic approach was adopted, in which the transcriptome of earlystage canine OA was assessed in the coronoid process samples. The canine data generated were meta-analysed alongside published datasets from in vivo models of early-stage OA. These data were from rodent models of the disease. A panel of genes were identified as being associated with the early stage of the disease across multiple datasets. By immunoassay, synovial fluid was screened for pro-inflammatory cytokines and in affected canine joints, interleukin 8 was found to be increased. Three in vitro models (cytokine stimulation of monolayer cell cultures, cyclic compression of agarose embedded cells and impact loading of osteochondral cores) were refined through modification of their stimuli. An identified panel of differentially expressed genes were used to screen each model under different parameters. Hierarchical clustering analysis was used to cluster the panel of conditions so that those which most closely reflected the naturally occurring disease were selected for more detailed transcriptomic analysis by microarray. Chondrocytes and osteoblasts were stimulated with a range of cytokine conditions, using IL-1β and IL-8 based on use in the literature and immunoassay findings. Monolayers were stimulated for a range of times and conentrations with either a single stimulus or multiple cytokines in the medium. The cells responded differently to the cytokine stimulus, requiring different stimuli to most closely replicate the transcriptomic profile of the natural disease. Microarray profiling revealed that cytokine stimulation enriched genes associated with the extracellular matrix and the extracellular region in both cells types. For the cyclic compression model, cells were embedded in an agarose gel matrix and cyclically compressed for various time periods followed by various incubation periods after compression. Both chondrocytes and osteoblasts responded in a similar manner to the cyclic compression stimulus when a post loading incubation step was included to replicate the transcriptomic profile of the natural disease. Cyclic compression enriched gene clusters associated with response to oxidative stress and the extracellular matrix When osteochondral cores were harvested from joints and impacted to represent a traumatic injury, the model could not replicate the transcriptomic model of the natural disease, although increased sGAG release nitric oxide (NO) production was observed. Degradation of mRNA in both tissues was a feature of this model regardless of the loading condition, which precluded further analysis by microarray, but highlighted the significant limitations that were associated with this model. None of the three models tested could accurately reflect the transcriptomic changes of the early-stage OA phenotype in cartilage or bone. A unified model, combining cytokine stimulation with cyclic compression drove cells towards the diseased phenotype in bone. Inflammatory pathways were activated as well as the proteases MMP3 and MMP13. However, chondrocytes were seemingly unresponsive to the multifactorial model, and this will require further analysis. The chronic nature of OA makes it difficult to match in vitro models to the transcriptomic phenotype identified in naturally occurring OA, particularly with respect to the differential expression of structural genes which were identified in the naturally occurring disease but not the models. This work highlights the limitations of existing models, but proposes a validation process which can be used to direct invitro models towards the naturally occurring phenotype.