The Antiepilptic drugs (AEDs) phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), tri- and dimethadione (TMD and DMD) are known teratogens having a common malformation pattern in human and animal studies. This thesis was designed chiefly to test a hypothesis correlating the teratogenicity of these AEDs to episodes of pharmacologically induced embryonic arrhythmia and hypoxia-reoxygenation damage. Effects on the embryonic heart were studied both after maternal administration in mice and in mouse embryos cultured in vitro. Only AEDs, correlated with the same type of malformation as could be induced by episodes of interrupted oxygen supply to the embryo (e.g. cleft palate) caused concentration dependent bradycardia and arrhythmia. PHT and DMD had the highest potential and affected embryonic heart at clinically relevant concentration, followed by CBZ, TMD and PB. Valproate and vigabatrin not associated with hypoxia-related malformations caused neither arrhythmia nor severe bradycardia. The results showed that the embryonic heart is extremely susceptible to PHT and DMD only during a restricted period of development, between gestational days 9-13 (weeks 5-9 of human pregnancy).An observed genetic susceptibility to react with arrhythmia at low concentrations when exposed to PHT or to external stress, could explain why A/J strain of mice is more susceptible to develop cleft palate compared to other strains. High activities of reactive oxygen species (ROS) capturing antioxidant enzymes observed in untreated A/J embryos supported this assumption. The potential to cause embryonic arrythmia by an AED was related to the potential to inhibit the rapid component of the delayed rectifier potassium channel (I kr ).A marked I kr blocking activity (70%)of DMD in voltage clamping studies was observed. The I kr inhibition occurred at similar concentrations, which causes severe arrhythmia. The idea of a relation between teratogenicity and arrhythmia, resulting in ischemia followed by reperfusion and generation of ROS was supported by mechanistic studies. Pre-treatment with the spin-trapping agent PBN, which has the capacity to capture ROS, markedly reduced the incidence of PHT and DMD-induced cleft palate. In utero exposure to teratogenic doses of DMD and PHT resulted in hemorrhages in the embryonic palatal region. The same type of haemorrhage in the palatal region precedes orofacial clefts induced by episodic hypoxia.

This thesis is concerned with the synthesis and evaluation of regenerable chalcogen containing antioxidants. Variously substituted 2,3-dihydrobenzo[b]selenophene-5-ol antioxidants were evaluated in order to gain information about structure/reactivity-relationships. Within the series explored, the most regenerable unsubstituted compound inhibited lipid peroxidation for more than 320 minutes when assayed in a two-phase lipid peroxidation model in the presence of N-acetylcysteine (NAC). α-Tocopherol which could inhibit lipid peroxidation for 90 minutes under similar conditions was therefore easily outperformed. The antioxidant activity of the parent was also documented in an aqueous environment. The best catalyst quenched/inhibited ROS production by neutrophils and PMA-stimulated macrophages more efficiently than Trolox. In addition, over a period of seven days, no disruption in proliferation for the cell lines used was observed when exposed to our synthetic compound or Trolox at a concentration of 60 µM. 3-Pyridinols substituted with alkyltelluro groups in the ortho-position were more regenerable in the two-phase model than their corresponding para-substituted analogues in the presence of NAC and also inhibited autoxidation of styrene in a catalytic fashion in homogenous phase in the presence of N-tert-butoxycarbonyl cysteine methyl ester (LipCys), a lipid-soluble analogue of NAC. The best inhibitors quenched peroxyl radicals more efficiently than α-tocopherol. They could also catalyze reduction of organic hydroperoxides in the presence of thiols and therefore mimic the action of the glutathione peroxidase enzymes. Mechanisms for the catalysis are proposed. Octylthio, octylseleno and octyltelluro analogues of butylated hydroxyanisole (BHA) were synthesized and evaluated. Among these, the tellurium compound was superior to α-tocopherol in the presence of NAC both when it comes to quenching capacity and regenerability.  Organochalcogen substituent effects in phenolic compounds were studied by using EPR, IR and computational methods.

Simvastatin, a cholesterol lowering drug, has the capacity to stimulate bone formation along with having anti-inflammatory effects. Incorporating simvastatin to the calcium phosphate cement would result in slow release of the drug stimulating bone formation and by preventing a local inflammation and bone resorption. The main aim is to study and examine the inflammatory response towards calcium phosphate cements in vitro and compare it with cements incorporated with simvastatin.

The innate immune system is important in both acute and chronic infection. In this thesis, I investigated the effect of H. pylori infection on 1) DCs, key orchestrators of the immune system, and 2) autophagy, recently identified as an important component of innate immunity. I determined that H. pylori activates the STAT3 pathway in DCs, increasing DC maturation and inducing production of IL-10, IL-12p40 and TNF-α, without IL-12p70. This cytokine profile may favour an immunoregulatory response, promoting persistent H. pylori infection. In addition I determined that H. pylori’s VacA toxin induced autophagy, ROS production and Parkin aggregation which has been implicated in mediating autophagy in response to mitochondrial damage. Thus H. pylori alters these key effectors of innate immunity which may play a role in promoting its chronic infection and disease.

The innate immune system is important in both acute and chronic infection. In this thesis, I investigated the effect of H. pylori infection on 1) DCs, key orchestrators of the immune system, and 2) autophagy, recently identified as an important component of innate immunity. I determined that H. pylori activates the STAT3 pathway in DCs, increasing DC maturation and inducing production of IL-10, IL-12p40 and TNF-α, without IL-12p70. This cytokine profile may favour an immunoregulatory response, promoting persistent H. pylori infection. In addition I determined that H. pylori’s VacA toxin induced autophagy, ROS production and Parkin aggregation which has been implicated in mediating autophagy in response to mitochondrial damage. Thus H. pylori alters these key effectors of innate immunity which may play a role in promoting its chronic infection and disease.

El presente trabajo de tesis doctoral se ha centrado en la determinación de la función fisiológica de la proteína desacopladora mitocondrial UCP3 en relación con la apoptosis y las especies reactivas de oxígeno (ROS). UCP3 es un miembro de la familia de transportadores mitocondriales que se expresa preferencialmente en el músculo esquelético. UCP3 es una proteína homóloga a la termogenina o proteína desacopladota UCP1. Ésta se expresa exclusivamente en tejido adiposo marrón dónde desempeña una función esencial en la termogénesis adaptativa, desacoplando la respiración mitocondrial de la fosforilación oxidativa con la consiguiente generación de calor. Así, como homólogo de la termogenina UCP3 podría desarrollar una actividad desacoplante mitocondrial influenciando el metabolismo energético celular, sin embargo su función biológica exacta continúa siendo fruto de controversia. En un primer trabajo, demostramos como la expresión ectópica de UCP3 en células de riñón humanas (293 HEK) mediante un sistema de expresión inducible sensible a tetraciclina, es consistente con un papel desacoplante mitocondrial de la proteína en la mitocondria de estas células. Observamos que la expresión de UCP3 no induce apoptosis directamente pero incrementa la sensibilidad a un agente inductor de apoptosis dependiente de vías de activación mitocondriales como es la staurosporina. Además, la inducción de UCP3 amuenta la sensibilidad del MPTP (mitochondrial transition pore) al calcio, facilitando la apertura del poro lo que se considera un evento crucial en la apoptosis dependiente de regulación mitocondrial. Estos datos sugieren una función de UCP3 en la regulación de la actividad del MPTP y sugieren que la presencia de UCP3 en la mitocondria sensibiliza a las células a los estímulos pro-apoptóticos que dependen de vías mitocondriales. Tras la obtención de los mencionados resultados, procedimos a inducir la expresión ectópica de UCP3 in vivo utilizando vectores adenovíricos dirigidos al hígado de ratón. El hígado normal no expresa ninguna proteína desacoplante y constituye por tanto un entorno ideal para determinar la función de UCP3. Resultados preliminares sugieren que la expresión ectópica de UCP3 no altera significativamente a parámetros metabólicos como la producción de ROS, aunque hallamos una estimulación de la apertura del MPTP en respuesta al calcio. Estos resultados procedentes de los estudios in vivo corroboran nuestros resultados previos obtenidos en líneas celulares en cultivo.Se cree que las proteínas desacoplantes podrían controlar la producción de ROS gracias a su capacidad para promover un desacoplamiento suave en la membrana mitocondrial interna. Hemos estudiado la función de UCP3 en su entorno natural de expresión, la célula del músculo esquelético, concretamente en lo que hace referencia a la muerte celular programada o apoptosis y las especies reactivas de oxígeno. Se demuestra que la expresión e UCP3 en miotubos no disminuye la producción de ROS, sino que incluso se incrementa en respuesta a un agente inductor de ROS como es la staurosporina. Igualmente, encontramos una sobreestimulación de la producción de ROS en respuesta a elevadas concentraciones de ácidos grasos, una situación en la que se estimula la expresión y actividad de las proteínas desacoplantes. Las ceramidas son productos secundarios del acúmulo de ácidos grasos capaces de inhibir la actividad de la cadena respiratoria mitocondrial. Los desacoplantes químicos pueden incrementar la producción de ROS en aquéllas situaciones en que la cadena respiratoria se encuentra inhibida. Pensamos que la sobre-inducción de ROS que observamos en presencia de UCP3 se deba a la acción conjunta de la proteína las ceramidas sobre la actividad respiratoria. Concluimos que UCP3 se comporta en los miotubos de forma compatible con una actividad desacoplante, promoviendo una sensibilización a la producción de ROS en respuesta a ciertos estímulos de estrés, como pueden ser un agente pro-apoptótico o elevadas concentraciones de ácidos grasos. Además, UCP3 promueve una sensibilización de la célula a la muerte programada inducida por estímulos dependientes de regulación mitocondrial. El análisis de la función de UCP3 en su entorno natural de expresión, el músculo esquelético, supuso además una profunda caracterización del comportamiento de la célula muscular ante los estímulos pro-apoptóticos. / My work of thesis has been focused on determining the role of mitochondrial uncoupling protein 3 (UCP3) in relation to apoptosis and reactive oxygen species (ROS). The mitochondrial uncoupling protein-3 is a member of the mitochondrial carrier protein family that is preferentially expressed in skeletal muscle. As a homologue of the thermogenic brown fat uncoupling protein-1, it possesses a mitochondrial uncoupling activity and thus can influence cell energy metabolism but its exact biological function remains unclear. We have previously proved that the induced expression of uncoupling protein-3 in the mitochondria of 293 cells using a tetracycline-inducible system was in agreement with its expected physiological behavior as an uncoupling protein (UCP). We observed that Uncoupling protein-3 expression did not cause apoptosis per se but increased the responsiveness of the cells to a mitochondrial apoptotic stimulus such as staurosporine, an apoptosis-inducer dependant on mitochondrial pathways. Moreover, the induction of uncoupling protein-3 increased the sensitivity of mitochondria to open the permeability transition pore (MPTP) in response to calcium, a well-known inducer of its opening which is a critical event in mitochondrial-driven apoptosis. This suggested for first time a role for UCP3 in cthe regulation of this channel activity. We could conclude that the presence of uncoupling protein-3 in mitochondria sensitizes cells to apoptotic stimuli involving mitochondrial pathways. Then, we induced in vivo ectopic expression of UCP3 in the liver of mice, using an adenoviral vector. Although preliminary, our results suggest that ectopic UCP3 expression is not significantly affecting to metabolic parameters or ROS production, but it is, as it happened in 293 cells, enhancing sensitivity of MPTP to its opening, corroborating our previous results. We are now characterizing the biochemical behaviour of UCP3 in the liver to finish a manuscript compelling all these results.Uncoupling proteins are thought to control ROS production due to their capacity to promote a mild uncoupling of the inner mitochondrial membrane. We have been studying UCP3 function in its natural environment of expression, the skeletal muscle cell. We have proved that UCP3 expression in myotubes does not decrease ROS production even in response to ROS-inducing agents such as staurosporine. In fact, it promotes enhanced ROS production in response to high fatty acid concentration. Ceramides are secondary products of fatty acid accumulation and can inhibit respiratory chain activity. Chemical uncouplers are known to increase ROS production in situations in which respiratory chain is inhibited. We are therefore currently exploring the possibility that ROS over-production could be due to the overlapping action of ceramides and UCP3.As a whole, we have demonstrated an involvement of UCP3 in apoptosis regulation and in the modulation of ROS production besides of performing a deep characterization of apoptosis responsiveness within skeletal muscle cell.

Generation of reactive oxygen species (ROS) is an important view point to evaluate heavy metal toxicity and resistance in plants. Arabidopsis thaliana is a fully sequenced model plant, and the characteristic between ecotypes due to adaptation towards varied environment can be used as a material for comparing physiological differences. In this experiments, two ecotypes of A. thaliana: Columbia (Col-0) and Landsberg erecta (Ler-0) is observed for the roots growth inhibition, plasma membrane integrity and lipid peroxidation after treated with different concentration of HgCl2 (0, 2, 4, 8 £gM), in attempt to compare the anti-oxidation defensive mechanism of two ecotypes and understand mercury-induced oxidative stress. ROS and Ca2+ generation is determined with CM-H2DCF-DA and Oregon Green 488 BAPTA-1 is under confocal microscopy. Some anti-oxidant enzymes such as superoxide dismutase (SOD EC 1.15.1.1), peroxidase (POD EC1.11.1.7) and ascorbate peroxidase (APX EC 1.11.1.11) are examined for the activity under protein gel electrophoresis. Experiment results showed that mercury-induced inhibition of root growth is more significant in Ler-0. ROS in roots of both ecotypes shows different trends under 8 £gM HgCl2 , however increment of ROS level below 4 £gM HgCl2 ; Ca2+ shows the similar result as ROS. Activity of SOD isoforms reached a peak at 2-4 £gM HgCl2. Expression of POD is correlated to the mercury concentration in both ecotypes. There are two types of APXs expression, one decreased as mercury concentration increased, another increased under 2 £gM HgCl2 and decreased as the concentration getting higher. According to the observation on expression of ROS generations and anti-oxidation system, we speculated that tolerance of Ler-0 towards mercury is weaker than Col-0. The results can be used as a basis for further discussion on influence of mercury towards different anti-oxidation enzymes and the signaling pathways.

La protéine UCP2 (UnCoupling Protein 2) appartient à la famille des transporteurs de la membrane interne de la mitochondrie. Son expression est restreinte à certains tissus comme la rate, l'estomac ou l'intestin. Au niveau cellulaire, UCP2 est particulièrement présente dans les macrophages où elle régule la production de radicaux libres (ROS). L'analyse des souris Ucp2-KO a montré qu'elles survivent mieux à une infection par le parasite Toxoplasma gondii que les animaux sauvages grâce à des macrophages superactifs en terme de production de ROS. Par ailleurs, dans le modèle murin de l'athérosclérose humaine, les souris Ucp2-KO développent des plaques athéromateuses plus instables et plus larges, présentant une forte accumulation de macrophages et des dégats importants liés au monoxyde d'azote (NO). <br />Au cours de ma thèse, nous avons cherché à approfondir les connaissances sur le rôle physiologique d'UCP2 ainsi que sur son activité biochimique.<br />Nous avons démontré que la diminution rapide d'UCP2 en réponse au LPS potentialise l'activation des MAPK dans les macrophages. La mitochondrie via UCP2 est ainsi au coeur d'une boucle d'amplification du signal impliquant la modulation des ROS mitochondriaux. Par conséquent, la signalisation et la vitesse d'activation des macrophages Ucp2-KO est accélérée, conduisant à une production accrue de NO et de cytokines.<br />La pertinance de ces résultats a ensuite été testée in vivo avec un volet infection et un volet auto-immunité. L'infection des souris par la bactérie Listeria monocytogenes a révélé une meilleure résistance des souris Ucp2-KO. Une production accrue de cytokines pro-inflammatoires chez les souris Ucp2-KO ainsi qu'un recrutement plus important de phagocytes au niveau de leur rate soulignent le rôle régulateur d'UCP2 sur l'immunité innée. En ce qui concerne, l'auto-immunité, l'induction expérimentale d'un diabète de type 1 est nettement accélérée chez les souris Ucp2-KO. L'analyse de ces souris montrent un rôle capital des macrophages dans le développement de la maladie grâce à leur forte capacité de production de cytokines et de NO.<br />L'activité biochimique d'UCP2, c'est-à-dire son activité de transport, a également été abordée. La glutamine est un inducteur spécifique de l'expression d'UCP2. Par conséquent, la comparaison du métabolisme de la glutamine dans les macrophages Ucp2-KO et Ucp2-WT a démontré que l'expression d'UCP2 est requise pour une oxydation correcte de la glutamine.<br />Enfin, grâce à la disponibilité de génomes complets de nombreuses espèces, l'étude phylogénomique des UCP a permis de tracer une histoire de l'évolution des UCP de mammifères et aviaire.<br />Nos études ont mis en évidence la participation d'UCP2 au métabolisme des macrophages. L'altération de celui-ci influe sur la signalisation et l'activité des cellules. Une meilleure compréhension de la fonction d'UCP2 et du métabolisme des cellules immunitaires pourrait ouvrir de nouvelles perspectives thérapeutiques.

Veränderungen im zellulären Oxidationszustand durch reaktive Sauerstoffspezies (ROS) sind in biochemischen Signalwegen, aber auch an der Entstehung von neurodegenerativen und anderen neuropathologischen Krankheiten beteiligt. Genetisch kodierte Redoxfarbstoffe haben sich in jüngerer Zeit als deutlich überlegen gegenüber der herkömmlichen Redox-Bildgebung gezeigt, um auch dynamische und quantitative Messungen des Redox-Status in komplexeren und adulten Organismen durchführen zu können. In dieser Arbeit wurde ein neues transgenes Mausmodell mit zytosolischer Expression des optischen Redox-Indikator roGFP1 in Neuronen unter der Kontrolle des Thy1-Promotors (C57BL/6-TG(Thy1.2-roGFP1c)) eingehend charakterisiert. Die transgenen Tiere zeigen einen mit verschiedenen Verhaltenstest und der Überprüfung verschiedener relevanter physiologischer Parameter sichergestellten gesunden Phänotyp. Bei an akuten Hirnschnitten und formalinfixierten Proben durchgeführten Messungen mit 2-Photonen-Laser-Scanning-Mikroskopie und herkömmlicher Fluoreszenzmikoskopie konnte eine kräftige Expression von roGFP1 vor allem in der CA1-Region, aber auch cortikal sowie im Hirnstamm gefunden werden. Durch Gegenfärbung mit fluoreszenmarkierten anti-NeuN-Antikörpern wurde eine extraneuronale Expression im Gehirn ausgeschlossen. Die Baseline der roGFP1-Antwort und Veränderungen im Oxidationszustand durch Inkubation von akuten Hirnschnitten mit Oxidations- und Reduktionsmitteln und unter Anoxie wurden dynamisch in Echtzeit mittels Fluoreszenzmikroskopie im Hippokampus und Neocortex bestimmt. Daraus kann geschlossen werden, dass das untersuchte C57BL/6-TG(Thy1.2-roGFP1c) Mausmodell funktionelle Redox-Indikatoren in ausreichendem Maße in großen Teilen des Gehirns exprimiert. Die Verfügbarkeit dieser Mäuse sollte sich als wichtig für das tiefergehende Verständnis der Rolle reaktiver Sauerstoffspezies und damit einhergehender Veränderungen des Redox-Status sowohl in der physiologischen Kontrolle der Zellfunktion als auch in neuropathologischen Prozessen erweisen.

In this thesis I develop a critical theory of public participation and deliberation in post-communist Romania. Primarily, this theory is developed from a critical assessment of two intermediate-level Romanian institutions: the deliberative system that has formed to debate the authorisation of a proposed gold mining project in Roșia Montana, Romania and the legitimacy claims of the series of hybrid forums that were convened as part of that project's Environmental Impact Assessment (EIA). The hybrid forum legitimacy claims were assessed based on a standard of hybrid forum legitimacy developed in this thesis from deliberative democracy theory and practice. Grounded in new institutionalism theory, this thesis describes the historical and contemporary socio-economic conditions that have shaped Romanian public participation and deliberation.