Dissertations / Theses on the topic 'Activation barrier'

T cells recognizing myelin auto-antigens penetrate into the CNS to induce inflammatory autoimmune disease following complex sequential interactions with individual components of the vascular blood-brain barrier (BBB), particularly endothelial cells, and perivascular phagocytes. To determine the functional consequences of these processes, two-photon intravital imaging was performed to compare the behavior of three myelin-specific GFP-expressing T cell lines with different potentials for transferring Experimental Autoimmune Encephalomyelitis. Imaging documented that, irrespective of their pathogenic potential, all T cell lines reached the CNS and interacted with vascular endothelial cells indistinguishably, crawling on the luminal surface, preferably against blood flow, before crossing the vessel wall. In striking contrast, after extravasation the T cell motility and their interactions with perivascular antigen presenting cells (APCs) varied dramatically. While highly encephalitogenic T cells showed a low motility, made stable contacts with local APCs and became activated, the corresponding contacts of weakly encephalitogenic T cells remained short, their motility high and their activation marginal. Supplying auto-antigen, via either local injection or by transfer of antigen-pulsed meningeal APCs, lowered their motility and prolonged the contact duration of weakly encephalitogenic T cells to values characteristic for highly pathogenic ones. Only after exogenous antigen supply, the weakly encephalitogenic T cells became activated, infiltrated the CNS parenchyma, and triggered clinical EAE, suggesting that the strength of the antigen-dependent signals received by immigrating effector T cells from leptomeningeal APCs is crucial for their pathogenic effect within the target tissue. To directly correlate the activation of encephalitogenic T cells with their dynamic behavior in the CNS, a truncated fluorescent derivative of nuclear factor of activated T cells (NFAT) was introduced as a real-time activation indicator. Two-photon imaging documented the activation of the auto-reactive T cells extravasated into the perivascular space, but not within the vascular lumen. Activation correlated with reduced T cell motility, and it was related to contacts with the local APCs. However, it did not necessarily lead to a long-lasting arrest, as individual, activated T cells SUMMARY 2 were able to sequentially contact other APCs. A spontaneous cytosol-nuclear translocation of the marker was noted only in T cells with a high pathogenic potential. The translocation implied the presentation of an auto-antigen, as the weakly pathogenic T cells, which remained silent in the untreated hosts, were activated upon the instillation of exogenous auto-antigen. It is proposed here that the presentation of local auto-antigen by BBB-associated APCs provides stimuli that guide autoimmune T cells to the CNS destination and enable them to attack the target tissue. In addition, a theoretical, physicist approach was used for modeling T cell activation in the leptomeningeal space. Assuming that T cells have evolved to gain their activation signal in a way that is energetically optimal for them, two possible scenarios for T cell activation were compared. The first one assumes that, after finding an APC presenting the epitope of interest, the T cell will stop and interact with the APC until it becomes fully activated. The second model considers the possibility that a T cell can accumulate activation signals from different APCs while scanning them without stopping, until a certain threshold is exceeded and the T cell becomes activated. Using this approach, it is proposed that the T cells in EAE are more likely to become activated following the first scenario. However, in a more natural environment such as a lymph node, the second scenario could give them some advantages

Voltage-gated ion channels play fundamental roles in neural excitability, they are for instance responsible for every single heart beat in our bodies, and dysfunctional channels cause disease that can be even lethal. Understanding how the voltage sensor of these channels function is critical for drug design of compounds targeting neuronal excitability. The opening and closing of the pore in voltage-gated potassium (Kv) channels is caused by the arginine-rich S4 helix of the voltage sensor domain (VSD) moving in response to an external potential. In fact, VSDs are remarkably efficient at turning membrane potential into conformational changes, which likely makes them the smallest existing biological engines. Exactly how this is accomplished is not yet fully known and an area of hot debate, especially due to the lack of structures of the resting and intermediate states along the activation pathway. In this thesis I study how the VSD activation works and show how toxic compounds modulate channel gating through direct interaction with these quite unexplored drug targets. First, I show that a secondary structure transition from alpha- to 3(10)-helix in the S4 helix is an important part of the gating as this helix type is significantly more favorable compared to the -helix in terms of a lower free energy barrier. Second, I present new models for intermediate states along the whole voltage sensor cycle from closed to open and suggest a new gating model for S4, where it moves as a sliding 3(10)-helix. Interestingly, this 3(10)-helix is formed in the region of the single most conserved residue in Kv channels, the phenylalanine F233. Located in the hydrophobic core, it directly faces S4 and creates a structural barrier for the gating charges. Substituting this residue alters the deactivation free energy barrier and can either facilitate the relaxation of the voltage sensor or increase the free energy barrier, depending on the size of the mutant. These results are confirmed by new experimental data that supports that a rigid ring at the phenylalanine position is the rate-limiting factor for the deactivation gating process, while the activation is unaffected. Finally, we study how the activation can be modulated for pharmaceutical reasons. Neurotoxins such as hanatoxin and stromatoxin push S3b towards S4 helix limiting S4's flexibility. This makes it harder for the VSD to activate and might explain the stronger binding affinities in resting state. All these results are highly important both for the general topic of biological macromolecules undergoing functionally critical conformational transitions, as well as the particular case of voltage-gated ion channels where understanding of the gating process is probably the key step to explain the effects of mutations or drug interactions.

QC 20121115

Hartree-Fock, Moller-Plesset, and density functional theory calculations have been carried out using 6-31+G(d), 6-31+G(d,p) and 6-31++G(d,p) basis sets to study the properties of low-barrier or short-strong hydrogen bonds (SSHB) and their potential role in enzyme-catalyzed reactions that involve proton abstraction from a weak carbon-acid by a weak base. Formic acid/formate anion, enol/enolate and other complexes have been chosen to simulate a SSHB system. These complexes have been calculated to form very short, very short hydrogen bonds with a very low barrier for proton transfer from the donor to the acceptor. Two important environmental factors including small amount of solvent molecules that could possibly exist at the active site of an enzyme and the polarity around the active site were simulated to study their energetic and geometrical influences to a SSHB. It was found that microsolvation that improves the matching of pK as of the hydrogen bond donor and acceptor involved in the SSHB will always increase the interaction of the hydrogen bond; microsolvation that disrupts the matching of pKas, on the other hand, will lead to a weaker SSHB. Polarity surrounding the SSHB, simulated by SCRF-SCIPCM model, can significantly reduce the strength and stability of a SSHB. The residual strength of a SSHB is about 10--11 kcal/mol that is still significantly stable compared with a traditional weak hydrogen bond that is only about 3--5 kcal/mol in any cases. These results indicate that SSHB can exist under polar environment. Possible reaction intermediates and transition states for the reaction catalyzed by ketosteroid isomerase were simulated to study the stabilizing effect of a SSHB on intermediates and transition states. It was found that at least one SSHB is formed in each of the simulated intermediate-catalyst complexes, strongly supporting the LBHB mechanism proposed by Cleland and Kreevoy. Computational results on the activation energy for catalyzed and uncatalyzed model reactions shows that strong hydrogen bonding between catalyst and the substrate at the transition state can significantly reduce the activation energy. This implies that LBHBs are possibly playing a crucial role in enzyme catalysis by supplying significant stabilizing energy to the reaction transition state.

Weaning is an important and complex step involving many stresses that interfere deeply with feed intake, gastro-intestinal tract (GIT) development and adaptation to the weaning diet in young pigs. The health of the pig at weaning, its nutrition in the immediate post-weaning period, and the physical, microbiological and psychological environment are all factors that interact to determine food intake and subsequent growth. GIT disorders, infections and diarrhoea increase at the time of weaning, in fact pathogens such as enterotoxigenic Escherichia coli (ETEC) are major causes of mucosal damage in post-weaning disease contributing to diarrhoea in suckling and post-weaned pigs. The European ban in 2006 put on antibiotic growth promoters (AGP) has stimulated research on the mechanisms of GIT disorders and on nutritional approaches for preventing or reducing such disturbances avoiding AGPs. Concerning these aspects here are presented five studies based on the interplay among nutrition, genomic, immunity and physiology with the aim to clarify some of these problematic issues around weaning period in piglets. The first three evaluate the effects of diets threonine or tryptophan enriched on gut defence and health as possible alternatives to AGP in the gut. The fourth is focused on the possible immunological function related with the development of the stomach. The fifth is a pilot study on the gastric sensing and orexygenic signal given by fasting or re-feeding conditions. Although some results are controversial, it appears that both tryptophan and threonine supplementation in weaning diets have a preventive role in E.coli PWD and favorable effects in the gut especially in relation to ETEC susceptible genotype. While the stomach is believed as almost aseptic organ, it shows an immune activity related with the mucosal maturation. Moreover it shows an orexygenic role of both oxyntic mucosa and pyloric mucosa, and its possible relation with nutrient sensing stimuli.

One of the severe complications of a Plasmodium falciparum infection is cerebral malaria (CM). CM is characterised by the accumulation of mature infected red blood cells (RBC) in the brain microvasculature. One of the consistent detrimental effects of sequestration is the breakdown of the blood-brain barrier (BBB), often with a fatal outcome in children in endemic areas. This study investigates the mechanisms underlying BBB breakdown secondary to sequestration, using immortalised human brain microvascular endothelial cells (tHBEC) as an in-vitro model of BBB and ITG-strain Plasmodium falciparum. First, the tHBEC monolayer was co-cultured with Plasmodium falciparum infected red blood cell (PRBC) or uninfected red blood cells (uRBC) control for 20 hours and the supernatant was recovered for subsequent analysis. The co-culture supernatants showed upregulation of inflammatory mediators (MCP-1 and IL-8) and a member of metalloproteases (ADAMTS-1, ADAMTS-4, MMP-2 and MMP-9) in the PRBC-tHBEC co-culture supernatants. The PRBC-tHBEC co-culture supernatants induced loss of endothelial cell monolayer integrity, represented by real time reduction in the transendothelial electrical resistance, measured using Electrical Cell-Substrate Impedance Sensing (ECIS™). The same supernatants also increased the permeability of tHBEC monolayer to the fluorescently labelled 40 kDa dextran showing leakage across the tHBEC monolayer. Interestingly, the loss of barrier function of tHBEC monolayer is partially inhibited by the addition of protease inhibitors GM6001 and rhTIMP-3. Prolonged exposure to PRBC-tHBEC co-culture supernatants reduced the level of vinculin. This study demonstrates that the interactions between PRBC and tHBEC induces activation of tHBEC and the release of proteases that contribute to BBB breakdown in CM, and could be a potential drug target for adjunct therapy in CM.

La barrière hémato-encéphalique (BHE) représente la principale interface d'échange moléculaire entre la circulation sanguine et le système nerveux central (SNC), où elle joue un rôle essentiel sur le contrôle du passage bidirectionnel de composés endogènes et exogènes. À la BHE, la P-glycoprotéine (P-gp) et Breast Cancer Resistance Protein (BCRP) sont les transporteurs d’efflux ABC les plus importants, empêchant l'entrée de composés toxiques, des médicaments et des xénobiotiques circulant dans le sang dans le cerveau. Il y a un intérêt croissant pour la compréhension des mécanismes moléculaires sous-jacents à la modulation de l’expression et de la fonction de la P-gp et BCRP, afin de pouvoir contrôler l'accumulation de substances neurotoxiques dans le SNC et de surmonter les phénomènes de pharmaco-résistance. Des études récentes ont montré que la morphine, elle-même un substrat de la P-gp, est impliquée dans l’augmentation de l'expression de la P-gp, qui peuvent contribuer à sa faible pénétration dans le cerveau et pour le développement de la tolérance. Cependant, le mécanisme sous-jacent à l’induction de la P-gp par la morphine, bien comme son rôle sur l'expression de BCRP était inconnu. Des rats ont été utilisés comme modèle animal pour l'étude de l'amplitude et la cinétique de la modulation de la P-gp et Bcrp à la BHE, après un traitement morphinique subchronique, en utilisant un protocole d’escalade de doses. Des microvaisseaux cérébraux isolés ont été utilisés comme modèle pour étudier la BHE, et les contenus en P-gp et Bcrp après le traitement in vivo, tandis que la lignée cellulaire hCMEC/D3 a parfois été utilisé pour des études complémentaires. Nos résultats ont montré qu’un régime subchronique de traitement à la morphine pendant 5 jours a induit la P-gp et Bcrp 12 à 24 heures après la dernière dose de morphine, un effet qui n'a pas été enregistrée lors des précédentes temps de sacrifices des animaux, ni avec une traitement aigue à la morphine. Le traitement des animaux avec un antagoniste de du récepteur glutamatergique NMDA, ou avec un inhibiteur de la COX-2 a aboli l’induction protéique de la P-gp et Bcrp par la morphine-subchronique, ce qui suggère que les deux facteurs sont impliqués dans l’up-régulation morphine-dépendante de la P-gp et BCRP. Sachant que l’induction a été enregistrée seulement à partir de 12h après la dernière dose de morphine, nous avons examiné si elle était un effet direct de l'exposition continue à la morphine, ou plutôt une conséquence du sevrage à la morphine développé après l'arrêt du traitement. Les rats ont été traités soit avec une perfusion constante de morphine (5 jours), soit avec deux schémas chroniques de morphine lorsque le sevrage a été précipité par l'administration de naloxone: un régime de doses croissantes (5 jours) ou un régime de doses constantes de morphine. La perfusion en continue de morphine n'a pas changé les niveaux de P-gp et Bcrp dans les microvaisseaux cérébraux de rat, et du coup n'a pas une conséquence directe sur la cascade de régulation de ces transporteurs à la BHE. Le sevrage provoqué par la naloxone a augmenté les niveaux d’ARNm pour le Mdr1a et Bcrp, mais l'expression et de l'activité protéiques sont restées inchangées après l'administration de naloxone. Cette disparité peut être dû soit à un effet de la régulation post-traductionnelle, soit à l’action de la naloxone dans des récepteurs non-opioïdes, qui peut entraver l’induction de la P-gp et Bcrp. Par la suite, on a fait un large screening de l'expression de plusieurs récepteurs de neurotransmetteurs chez la BHE de rat, beaucoup d'entre eux impliqués dans la signalisation inflammatoire, et qui peut jouer un rôle dans la modulation de ces transporteurs ABC. (...)
The blood-brain barrier (BBB) is the main interface of molecular exchange between the bloodstream and the central nervous system (CNS), where it plays an essential role on the control over the bi-directional passage of endogenous and exogenous compounds. At the BBB, P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are the most important ABC drug efflux transporters preventing the entry into the brain of toxic compounds, drugs and xenobiotics circulating in the blood. There is increasing interest in understanding the molecular mechanisms underlying the modulation of P-gp and BCRP expression and function in order to control CNS accumulation of neurotoxicants and to overcome pharmacoresistance phenomena. Recent studies showed that morphine, itself a substrate of P-gp, is implicated in the up-regulation of P-gp expression, which may contribute to its poor brain penetration and tolerance. However, it was unknown the mechanism underlying P-gp induction by morphine and its role on BCRP expression. Rats were used as an animal model for the study of the amplitude and the kinetics of the modulation of P-gp and Bcrp expressions at the BBB following a subchronic morphine treatment, in an escalating morphine dose regimen. Freshly isolated rat brain microvessels were used as BBB model to study P-gp and Bcrp contents following the in vivo treatment, while the hCMEC/D3 cell line was occasionally used for complementary studies. Our results demonstrated that a 5-day subchronic morphine regimen up-regulated both P-gp and Bcrp 12 to 24h after the last dose of morphine, which was not registered at earlier time-points of animal sacrifice, nor with a single dose of morphine. The animal treatment with a glutamatergic NMDA receptor antagonist, or a COX-2 inhibitor abolished the subchronic morphine-induced P-gp and Bcrp protein up-regulation, 24h after the last dose of morphine, suggesting that both are implicated in the morphine-dependent P-gp and Bcrp up-regulation. Since the registered up-regulation only occurred from 12h after the last dose of morphine-onwards, we investigated whether it was a direct effect of continued exposure to morphine, or rather a consequence of the morphine withdrawal developed after discontinuation of treatment. Rats were treated either with a constant morphine infusion (5 days), or two chronic morphine regimens where withdrawal was precipitated by naloxone administration: an escalating dose (5 days) or a constant dose morphine regimen followed by a withdrawal period (2 days) and resume of the treatment for 3 additional days. Continuous i.v. morphine did not change P-gp and Bcrp levels in rat brain microvessels, it does not have a direct consequence on the cascade of regulation of these transporters at the BBB. Naloxone-precipitated withdrawal after escalating or chronic morphine dose regimen increased Mdr1a and Bcrp mRNA levels, but protein expression and activity remained unchanged after naloxone administration. This latter result discrepancy may be due to posttranslational regulation or naloxone action at non-opioid receptors hampering P-gp and Bcrp up-regulation. Subsequently, we did a large screening of the expression of several neurotransmitter receptors at the rat BBB, many of them implicated in the inflammatory cell-cell signaling, and which may have a role in the modulation of these ABC transporters. Also, we compared two different approaches of isolation of rat brain microvessels, mechanical dissection and enzymatic digestion, to assess which yield the purest microvessel fraction for the BBB study. The enzymatic digestion provided the highest enrichment of endothelial cells and pericytes, and the least contamination with astrocyte and neuron markers. (...)
A barreira hemato-encefálica (BHE) representa a principal interface entre a corrente sanguínea e o sistema nervoso central (SNC), desempenhando um papel essencial no controlo da passagem sangue-cérebro de diversos compostos endógenos e exógenos. A glicoproteina P (P-gp) e a proteína de resistência ao cancro da mama (BCRP) são os principais transportadores de efluxo da família ABC presentes ao nível da BHE, limitando a passagem cerebral de compostos tóxicos, fármacos e xenobióticos circulantes na corrente sanguínea. Actualmente, regista-se um crescente interesse na comunidade científica para a melhor compreensão dos mecanismos moleculares subjacentes à modulação quer da expressão quer da função da P-gp e BCRP, no sentido de desenvolver medidas mais eficazes quer para prevenção da acumulação de compostos neurotóxicos no SNC, quer para superar fenómenos de farmacorresistência associados à terapêutica. Estudos recentes evidenciam que a morfina, por si só um substrato da P-gp, está envolvida na indução da expressão da P-gp, o que poderá contribuir para a sua menor penetração cerebral, bem como para o desenvolvimento de tolerância. No entanto, não se conhece o mecanismo subjacente a tal indução da P-gp pela morfina, nem o seu eventual papel na expressão da BCRP. Com efeito, na condução da presente dissertação, realizamos um estudo da amplitude e a cinética da regulação da expressão da P-gp e BCRP ao nível da BHE na sequência de um tratamento subcrónico com morfina, em regime de doses crescentes, usando o rato como modelo animal. Para o efeito, foram isolados os capilares cerebrais dos animais sujeitos a tratamento, in vivo, enquanto que a linha celular hCMEC/D3 foi ocasionalmente utilizada para estudos complementares. Os nossos resultados demonstraram que um tratamento subcrónico com morfina (5 dias) foi capaz de induzir tanto a P-gp como a Bcrp 12 a 24 horas após a última dose de morfina administrada, mas não para tempos de sacrifício anteriores, bem como tal indução não foi registada quando a morfina foi administrada de forma aguda. O tratamento animal com um antagonista do receptor glutamatérgico NMDA, ou com um inibidor da COX-2 anulou este efeito de indução da P-gp e Bcrp pela administraçãosubcrónica de morfina, o que sugere o envolvimento destes dois componentes na indução da P-gp e Bcrp dependente da morfina. Uma vez que este aumento da expressão só surgiu a partir de 12h após a última dose de morfina, decidimos investigar se tal seria um efeito direto da exposição continuada à morfina, ou por outro lado, uma consequência do síndrome de abstinência à morfina, desenvolvido após a descontinuação do tratamento. Desta forma, os animais foram tratados por um lado com uma infusão contínua de morfina (5 dias), ou sujeitos a dois diferentes regimes de exposição crónica à morfina, após os quais o síndrome de abstinência foi provocado pela administração de naloxona. A administração de morfina em contínuo, via i.v., não alterou os níveis de P-gp e BCRP nos capilares cerebrais de rato, o que indica a ausência de uma consequência directa da morfina na cascata de regulação destes transportadores ao nível da BHE. O síndrome de abstinência opióide provocado pela naloxona aumentou os níveis de mRNA Mdr1a e Bcrp, mas tanto a expressão e atividade proteicas mantiveram-se inalteradas após a administração de naloxona. Esta discrepância de resultados pode-se dever ou a um regulamento pós-translacional, ou a uma acção inespecífica da naloxona em receptores não opiáceos, impedindo a indução da P-gp e Bcrp. Num outro estudo, foi feito um screening da expressão de vários receptores de neurotransmissores na BHE de rato, muitos deles envolvidos na sinalização célula-célula em processos inflamatórios, e que podem ter um papel na modulação destes transportadores ABC. (...)

Chez les individus infectés par le VIH on observe une augmentation de l’inflammation et de l’activation immunitaire (AI) qui est associée à la progression vers le SIDA. Même en présence de traitements antirétroviraux, les niveaux d’AI restent élevés et sont associés à une morbidité et une mortalité accrues. Les mécanismes moléculaires derrière l’AI ne sont pas bien caractérisés. L’activation des macrophages (Mφ ) et monocytes et la translocation bactérienne peuvent jouer un rôle important. Contraire aux humains, et au model pathogène de l’infection (macaques, MAC), les hôtes naturels du SIV (Singes vertes d’Afrique, AGM), résoudraient l’inflammation aiguë, ils montrent un niveau plus bas de cytokines inflammatoires comme l’IL-1β et l’IL-18 et l’IA chronique est absente. L’IL-1β et l’IL-18 sont produits par le Mφ et les cellules épithéliales de l’intestine (IEC) auprès l’activation de l’inflammasome. Nous avons étudié l’activation de l’inflammasome chez les hôtes naturels, dans quel tissues ceci peut avoir lieu et s’il existe des différences entre les deux modelés d’infection. Pour cela, nous avons mesuré les niveaux plasmatiques de l’IL-1β et l’IL-18 au cours de l’infection ; nous avons analysé le marquage des Mφ , IEC et IL-18 par microscopie confocale; nous avons mis en place des essaies fonctionnels pour l’activation in vitro de l’inflammasome et nous avons développé les outils pour le phenotypage et l’isolation de Mφ et IEC du sang, du poumon, du LAB, des ganglions et de l’intestine. Nous avons montré que l’inflammasome est activé lors de l’infection par SIV dans les modèles pathogène et non-pathogène, en particulier dans l’intestine grêle. Notre étude indique une production plus notable d’IL-18 dans le jéjunum des MAC infectés en comparaison avec des AGM. Nous avons montré que l’inflammasome peut être activé dans les macrophages par des signaux de l’environnement présent lors de la translocation bactérienne et le stress cellulaire, comme le LPS et l’ATP. Nous avons montré de différences au niveau de régulation de la réponse liée à l’inflammasome. Les niveaux d’IL-18BP et l’IL-1RA, les antagonistes de l’IL-18 et l’IL-1β respectivement, sont été plus élevés chez les AGM que chez les MAC. On a trouvé une corrélation entre les niveaux de IL-18BP/IL-1Ra et les niveaux plasmatiques des agonistes chez les hôtes naturelles mais pas chez les MAC, ce qui est indicative de une régulation potentiellement plus efficace chez les AGM
Chronic immune activation drives progression toward AIDS in HIV infection and still remains in low levels in antiretroviral-treated patients increasing the risk of non-communicable diseases. Such non-AIDS co-mobility and mortality is associated with markers of monocyte/macrophage (Mφ ) activation and microbial translocation, but the molecular bases of this phenomenon remain unknown. In contrast to humans and pathogenic animal models of HIV (i.e. macaques, MAC), natural hosts of SIV (i.e. African Green Monkeys, AGM) quickly resolve SIV-induced inflammation and display lower levels of IL-1β and IL-18. IL-1β and IL-18 can be produced by Mφ or intestinal epithelial cells (IEC) upon inflammasome activation with potential multiple roles. Therefore, we studied whether the inflammasome activation upon SIV-infection occurs in natural hosts, in which tissues it might take place and if it differs between models. To do so, we measured plasmatic IL-1β and IL-18 levels along SIV-infection; we performed microscopy staining of Mφ , IEC and IL-18 in tissues, we set-up functional assays for inflammasome activation in-vitro and we developed tools for phenotyping and isolating Mφ and IEC from blood, lung, BAL, LN and gut. We showed inflammasome activation in vivo during pathogenic and non-pathogenic SIV infection evaluated by IL-18 in the gut of MAC and AGM, particularly in the small intestine, as well as by the levels of IL-18 and IL-1β in plasma. Our study indicated higher IL-18 production in the jejunum of SIV-infected MAC as compared to SIV-infected AGM. We showed that signals that might be in the environment during pathogenic SIVmac infection, in particular LPS and ATP as a result of microbial translocation and stress activate the inflammasome of MAC and AGM macrophages. We revealed differences at the level of the regulation between both models, observed by higher levels of IL-18BP and IL-1RA in AGM compared to MAC and correlations between IL-18, IL-1β and their respective antagonists only in AGM but not in MAC

Voltage-gated ion channels play fundamental roles in neural excitability and thus dysfunctional channels can cause disease. Understanding how the voltage-sensor of these channels activate and inactivate could potentially be useful in future drug design of compounds targeting neuronal excitability. The opening and closing of the pore in voltage-gated ion channels is caused by the arginine-rich S4 helix of the voltage sensor domain (VSD) moving in response to an external potential. Exactly how this movement is accomplished is not yet fully known and an area of hot debate. In this thesis I study how the opening and closing in voltage-gated potassium (Kv) channels occurs. Recently, both experimental and computational results have pointed to the possibility of a secondary structure transition from α- to 3(10)-helix in S4 being an important part of the gating. First, I show that the 3(10)-helix structure in the S4 helix of a Kv1.2-2.1 chimera protein is significantly more favorable compared to the α-helix in terms of a lower free energy barrier during the gating motion. Additional I suggest a new gating model for S4, moving as sliding 310-helix. Interestingly, the single most conserved residue in voltage- gated ion channels is a phenylalanine located in the hydrophobic core and directly facing S4 causing a barrier for the gating charges. In a second study, I address the problem of the energy barrier and show that mutations of the phenylalanine directly alter the free energy barrier of the open to closed transition for S4. Mutations can either facilitate the relaxation of the voltage-sensor or increase the free energy barrier, depending on the size of the mutant. These results are confirmed by new experimental data that supports that a rigid, cyclic ring at the phenylalanine position is the determining rate-limiting factor for the voltage sensor gating process.
QC 20110616

DDétermination des caractéristiques du silicium amorphe hydrogène par mesure et analyse des admittances de diodes Schottky fournies sur ce matériau: densité d'états en-dessous et au niveau de fermi avec section de capture efficace des électrons et énergie d'activation du matériel.

Timely activation of the rapid response team (RRT) depends on the nurse's willingness and ability to make a rapid decision. The practice-focused questions for this DNP project sought to identify barriers that contribute to delays in activating the RRT when needed in medical-surgical patients. The self-efficacy theory was the guiding theory and was used to examine self-confidence and performance along with Donabedian's health care model. Qualitative data were obtained through focus groups and identified 2 prominent thematic barriers among nurses with less than 3 years' experience: a lack of self-confidence and the of lack of knowledge and experience. Results of a chart review included 34 charts to determine if the RRT were called appropriately and were inconclusive. Finally, an 11 item survey with 9 demographic questions showed a statistically significant difference on the summed survey score between nurses with less than 3 years of experience and more tenured nurses, indicating a lack of perceived support, self-confidence, and knowledge among the nurses with less than 3 years of experience (Pearson chi square = 7.403 with 2 df and p = .025). Results were presented to leaders at the site and the recommendations resulting from these observations include the use of high-fidelity simulation education. Nurse educators and senior leadership from the medical surgical units agreed to accept the recommendations and proceed with developing an educational solution to address the barriers. Building knowledge, skills and self-confidence in nurses reduces the barriers to effective use of the RRT, and results in better outcomes for hospitalized medical-surgical patients, a positive social change.

Für Dihydropyrene ist die 6 pi Elektrozyklisierung für gewöhnlich schnell, wohingegen die Cycloreversion durch eine Aktivierungsbarriere im angeregten Zustand ineffizient wird. Die Substitution mit Donor- und Akzeptorgruppen erzeugt ein „push-pull“ System, welches eine bathochrome Verschiebung der Absorption in den tief roten Bereich (730 nm onset) zur Folge hat. Das „push-pull” System polarisiert den Dihydropyrenkern, was ein Absenken der Aktivierungsbarriere im angeregten Zustand zur Folge hat und in einer erhöhten Quantenausbeute resultiert. Es wird weiter gezeigt, wie dies in nicht-permanenter Art und Weise durch Katalyse vollzogen werden kann, indem eine protonierte Spezies mit einer geringeren Aktivierungsbarriere im angeregten Zustand gebildet wird. Da Dihydropyrene im sichtbaren Bereich absorbieren und im Allgemeinen als T-Typ negativ photochrom betrachtet werden, ist ein Schalten ohne die Verwendung von UV Licht möglich. Für die Substanzklasse der Azobenzole wird ein neuer aromatischer Substituent anstelle eines der Phenylreste untersucht. Die Bandenseparation von bis zu 80 nm erlaubt hohe photostationäre Zustände von ≈ 90 % für beide Richtungen. Die hohen Extinktionskoeffizienten von ≈ 20000 L mol 1cm 1 für das E Isomer, führen zu einer gesteigerten Absorption im Vergleich zu herkömmlichen Azobenzolen. Weiterhin kann die Löslichkeit der Verbindungen, durch die Wahl des Gegenions moduliert werden, was es ermöglicht in Tetrahydrofuran, Acetonitril, Wasser, sowie einer ionischen Flüssigkeit zu schalten. Mit höherer Polarität des Lösungsmittels wird die Absorptionsbande des E Isomers zu längeren Wellenlängen hin verschoben und die thermischen Rückreaktion beschleunigt. Die thermische Halbwertzeit der Rückreaktion kann zwischen 3 min und 13 h bei 25 °C eingestellt werden. Ebenso wurde auch das Schaltverhalten einer Azoniumspezies untersucht und eine erstaunlich lange thermische Halbwertzeit von > 2 min beobachtet.
For the dihydropyrene system 6 pi electrocyclization is usually fast, while the cycloreversion is inefficient, due to an activation barrier in the excited state. It is shown, how substitution with donor and acceptor moieties creates a push-pull system, causing a bathochromic shift of the absorption spectrum to the far red (730 nm onset). The push-pull system induces a dipole in the dihydropyrene, which lowers its excited state activation barrier and therefore increases the quantum yield of the cycloreversion. Further it is shown, how this can be performed in a catalytic fashion, where protonation leads to a species with a lower barrier in the excited state. As dihydropyrenes absorb in the visible and are considered as T-type negative photochromic, they can be switched without the use of UV-light. In case of the azobenzene class, a new aromatic substitute for one of the benzene rings is investigated and shows superior switching properties. Band separation of up to 80 nm is shown, along with high photostationary states ≈ 90% favoring each of the two switching directions. Interestingly, the extinction coefficient especially of the E isomer increases to ≈ 20000 L mol-1cm-1, dramatically enhancing the absorptivity compared to normal azobenzenes. Furthermore, the solubility can be tuned by proper choice of the cation, which is used to investigate solvent effects in nonpolar, polar, and protic (water) solvents as well as in an ionic liquid. With increasing polarity, the absorbance of the E isomer is shifted to longer wavelengths, which is accompanied by a reduced thermal half-life. The half-life of the thermal reverse reaction can be tuned from 3 min to 13 h at ambient temperature. As one of the derivatives is easily protonated, switching of the corresponding azonium species has also been investigated and an astoundingly long thermal half-life of > 2 min at room temperature has been observed.

Durant ces dernières années, l’apparition de nouvelles architectures (FDSOI, FinFETs ou NW-FETs) et l’utilisation de nouveaux matériaux (notamment SiGe) ont permis de repousser les limites des performances des dispositifs MOS et de contourner l’effet canal court inhérent à la miniaturisation des composants. Cependant, pour toutes ces nouvelles architectures, la résistance de contact se dégrade au fil des nœuds technologiques. Celle-ci dépend fortement de deux paramètres physiques : la concentration de dopants actifs proches de la surface du semi-conducteur et de la hauteur de barrière Schottky du contact siliciuré. De multiples procédés avancés ont été proposé pour améliorer ces deux paramètres physiques (pré-amorphisation, recuit laser, ségrégation de dopants, etc…). Afin d’optimiser les conditions expérimentales de ces nouvelles techniques de fabrication, il est primordial de pouvoir caractériser avec fiabilité leur impact sur les deux grandeurs physiques citées. Dans le cadre de cette thèse, deux thématiques dédiées à l’étude de chacun des paramètres sont abordées, explicitant les méthodes de caractérisation développées ainsi que des exemples concrets d’applications. La première partie concerne l’étude de la concentration de dopants actifs proches de la surface du semi-conducteur. Dans cet axe, nous avons mis en place une méthode d’Effet Hall Différentiel (DHE). Cette technique combine gravures successives et mesures par effet Hall conventionnel afin d’obtenir le profil de concentration de dopants actifs en fonction de la profondeur. Nous avons développé et validé une méthode de gravure chimique et de mesure électrique pour des couches ultra-minces de SiGe et de Si dopées. Les profils de concentration générés ont une résolution en profondeur inférieure à 1 nm et ont permis d’étudier de façon approfondie dans les premiers nanomètres proches de la surface de couches fabriquées grâce à des techniques d’implantation et de recuit avancées comme par exemple, la croissance en phase solide activée par recuit laser. La deuxième partie porte sur la mesure de hauteurs de barrière Schottky pour des contacts siliciurés. Durant cette étude, nous avons transféré une technique se basant sur des diodes en tête bêche pour caractériser l’impact de la ségrégation de différentes espèces à l’interface siliciure/semi-conducteur sur la hauteur de barrière Schottky d’un contact en siliciure de platine. Cette méthode de mesure associée à des simulations physiques a permis d’une part, d’extrairer avec fiabilité des hauteurs de barrières avec une précision de 10meV et d’autre part, d’effectuer une sélection des meilleures conditions de ségrégation de dopants pour la réduction de la hauteur de barrière Schottky. Pour conclure, ce projet a rendu possible le développement de méthodes de caractérisation pour l’étude de matériaux utilisés en nanoélectronique. De plus, nous avons pu apporter des éclaircissements concernant l’impact de techniques d’implantation ionique alternatives sur des couches de Si et SiGe ultrafines, et ce, dans le but de réduire la résistance de contact entre siliciure et semi-conducteur dans le module source-drain de transistors ultimes
During the past few decades, the emergence of new architectures (FDSOI, FinFETs or NW-FETs) and the use of new materials (like silicon/germanium alloys) allowed to go further in MOS devices scaling by solving short channel effect issues. However, new architectures suffer from contact resistance degradation with size reduction. This resistance strongly depends on two parameters: the active dopant concentration close to the semi-conductor surface and the Schottky barrier height of the silicide contact. Many solutions have been proposed to improve both of these physical parameters: pre-amorphisation, laser annealing, dopant segregation and others. In order to optimize the experimental conditions of these fabrication techniques, it is mandatory to measure precisely and reliably their impact on cited parameters.Within the scope of this thesis, two parts are dedicated to each lever of the contact resistance, each time precising the developed characterization method and concrete application studies. The first part concerns the study of the active dopant concentration close to the semi-conductor surface. In this axis, we developed a Differential Hall Effet method (DHE) which can provide accurate depth profiles of active dopant concentration combining successive etching processes and conventional Hall Effect measurements. To do so, we validated layer chemical etching and precise electrical characterization method for doped Si and SiGe. Obtained generated profiles have a sub-1nm resolution and allowed to scan the first few nanometers of layers fabricated by advanced ion implantation and annealing techniques, like solid-phase epitaxy regrowth activated by laser annealing. In the second part, we focused on the measurement of Schottky barrier height of platinum silicide contact. We transferred a characterization method based on back-to-back diodes structure to measure platinum silicide contacts with different dopant segregation conditions. The electrical measurements were then fitted with physical models to extract Schottky barrier height with a precision of about 10meV. This combination between measurements and simulations allowed to point out the best ion implantation and annealing conditions for Schottky barrier height reduction.To conclude, thanks to this project, we developed highly sensitive characterization methods for nanoelectronics application. Moreover, we brought several clarifications on the impact of alternative ion implantation and annealing processes on Si and SiGe ultra-thin layers in the perspective of contact resistance reduction in FDSOI source-drain module

Protein (mis)folding into highly ordered, fibrillar structures, amyloid fibrils, is a hallmark of several, mainly neurodegenerative, disorders. The mechanism of this supra-molecular self-assembly reaction, as well as its relationship to protein folding are not well understood. In particular, the molecular origin of the metastability of the soluble state of proteins with respect to the aggregated states has not been clearly established. In this dissertation, it is demonstrated, that highly accurate kinetic experiments, using a novel biosensing method, can yield fundamental insight into the dynamics of proteins in the region of the free energy landscape corresponding to protein aggregation. First, a section on Method development describes the extension and elaboration of the previously established kinetic assay relying on quartz crystal microbalance measurements for the study of amyloid fibril elongation (Chapter 3). This methodology is then applied in order to study in great detail the origin of the various contributions to the free energy barriers separating the soluble state of a protein from its aggregated state. In particular, the relative importance of residual structure, hydrophobicity (Chapter 4) and electrostatic interactions (Chapter 5) for the total free energy of activation are discussed. In the last part of this thesis (Chapter 6), it is demonstrated that this biosensing method can also be used to study the binding of small molecules to amyloid fibrils, a very useful feature in the framework of the quest for potential inhibitors of amyloid formation. In addition, it is shown that Thioflavin T, to-date the most frequently employed fluorescent label molecule for bulk solution kinetic studies, can in the presence of potential amyloid inhibitor candidates be highly unreliable as a means to quantify the effect of the inhibitor on amyloid formation kinetics. In summary, the work in this thesis contributes to both the fundamental and the applied aspects of the field of protein aggregation.

V mnoha oborech jsou stále využívána anorganická filtrační media založená na materiálech, jejichž výroba využívá primární suroviny. Jejich výroba je tedy energeticky náročná a v důsledku nákladná a neohleduplná k životnímu prostředí. Cílem této práce je ověřit možnost využití alkalicky aktivovaných materiálů na bázi sekundárních surovin, především vysokopecních strusek (BFS) a popílků z uhelných elektráren (FA), pro výrobu porézních médií schopných v budoucnosti nahradit keramické a jiné anorganické filtry. Výzkum je rozvinut skrze experimentální design založený na výpočetním schématu samostatně vyvinutém s pomocí programu MATLAB. Toto schéma počítá vhodná složení směsí na základě poměrů obsahu nejdůležitějších oxidů ve vstupních surovinách. Tak je zajištěno zohlednění proměnlivého složení vstupních surovin a práce je tím hodnotnější, že její výsledky jsou skrze početní nástroj zohledňující základní oxidové složení surovin zobecnitelné. Zároveň byly však pro srovnání a lepší názornost závislostí vlastností na složení navrhnuty a připraveny i série vzorků založené vždy pouze na jedné ze surovin. Z výsledků vyplývá, že pevnost vzorků z těchto směsí (vytvrzených 24 hodin při 70 °C) ve čtyřbodové ohybové zkoušce dle ČSN EN 12390 5 může přesáhnout 7,6 MPa. Dosažením co možná nejvyšší porozity však zákonitě negativně ovlivňuje pevnost materiálu a výsledný materiál tedy dosahuje pevnosti těsně nad hranicí 6,3 MPa. Výsledky obecně dokazují, že nejvíce je pevnost materiálů ovlivněna poměrem SiO2/Al2O3 a množstvím alkalického aktivátoru. Z výsledků vyplývá, že alkalicky aktivované materiály (AAM) na bázi strusky dosahují i více než dvojnásobné pevnosti analogických materiálů na bázi elektrárenského popílku. Velikost pórů materiálů připravených z tříděných surovin s velikostí zrna od desítek po lehce přes sto mikronů se ve většině případů pohybuje v rozmezí desetin ž jednotek mikronů, v případě výsledného materiálu je to pak přibližně 0,2 mikronu. Celková porozita lisovaných těles se pohybuje těsně pod 40 %, což je v tomto případě téměř dvojnásobek ve srovnání s totožnými materiály na bázi netříděných surovin. Výsledky rovněž ukazují, že materiály na bázi strusky vykazují nižší porozitu než ty na bázi popílku, což je patrně způsobeno rozdílnou morfologií částic obou materiálů – částice strusky jsou nepravidelně hranaté a částice popílku kulaté. V průběhu experimentální činnosti byla pozorována tvorba výkvětů u materiálů na bázi elektrárenských popílků. Pomocí Energo-disperzní spektroskopické analýzy (EDS) byly výkvěty identifikovány jako hydroxid sodný procházející karbonatací za účasti vzdušného CO2. Test permeability vyžadoval, kvůli velmi jemné povaze porézní struktury, přípravu asymetrických filtračních přepážek. Tyto přepážky dosáhli propustnosti 138 L/h.m2.bar pro vodu a 1320 L/h.m2.bar pro vzduch.

La neuroinflammation est un dénominateur commun de plusieurs troubles du système nerveux central. Les réactions inflammatoires sont souvent médiées par plusieurs voies de signalisation qui conduisent à l'ouverture de la barrière hémato-encéphalique. L'activateur tissulaire du plasminogène (tPA) est une serine protéase qui induit l'ouverture de la barrière hémato-encéphalique. Au cours des dernières années, il a également été montré que les récepteurs NMDA situés dans les cellules endothéliales peuvent jouer un rôle crucial dans la propagation de la réaction inflammatoire.Mon travail au cours de ma thèse a mis l'accent sur la découverte des mécanismes par lesquels le récepteur NMDA effectue une médiation de l'ouverture de la barrière hémato-encéphalique induite par le TPA. Dans notre première étude, nous montrons que les récepteurs NMDA endothéliaux sont des cibles thérapeutiques potentielles pour prévenir l'infiltration et l'inflammation des cellules immunitaires médiées par l'EAE. Nous montrons que l'anticorps monoclonal du récepteur NMDA spécifique à la souris, le Glunomab, pourrait protéger la barrière de la moelle épinière de dommages inflammatoires. Nous montrons également que les récepteurs NMDA sont exprimés en étroite association avec les protéines de jonction serrées dans les cellules endothéliales cérébrales. Dans notre deuxième étude, nous montrons pour la première fois que les récepteurs NMDA neuroendothéliaux peuvent présenter une action métabotropique lors de l'inflammation. Nous soulignons également que ces récepteurs sont en effet des récepteurs NMDA non conventionnels exprimant la sous unité GluN3A. En outre, nous rapportons que le tPA accélère l'ouverture de la barrière hémato-encéphalique en présence d'une agoniste rare de la glycine par un mécanisme dépendant de l'activation de RhoA. Les résultats de mon projet apportent une nouvelle vision du rôle des récepteurs NMDA métabotropiques dans les cellules endothéliales cérébrales. En outre, il fournit également des détails plus précis sur l'ouverture de la barrière hémato-encéphalique via l’activateur tissulaire du plasminogène
Neuroinflammation is a common denominator of several central nervous system disorders. Inflammatory reactions are often mediated by several signaling pathways which lead to the opening of the blood brain barrier. Tissue plasminogen activator (tPA) is a serine protease induces opening of the blood brain barrier. In recent years, it has also been shown that NMDA receptors located in endothelial cells can play a crucial role in propagation of inflammatory reaction. My doctoral study focused on the finding the underlying mechanisms of action(s) by which NMDA receptor mediates tPA induced opening of the blood brain barrier. In our first study we show that endothelial NMDA receptors are potential therapeutic targets to prevent EAE mediated immune cell infiltration and inflammation. We show that NMDA receptor specific mouse monoclonal antibody Glunomab could prevent the brain spinal cord barrier from inflammatory damage. We also show that NMDA receptors are expressed in close association of tight junction proteins in cerebral endothelial cells. In our second study, we show for the first time that, neuroendothelial NMDA receptors can exhibit metabotropic mode of action during inflammation. We also highlight that these receptors are indeed GluN3A expressing non-conventional NMDA receptors. In addition, we report that tPA accelerates the opening of blood brain barrier in presence of an uncommon agonist glycine by RhoA activation dependent mechanism.My project results provide a nouvelle insight for the role of metabotropic NMDA receptors in cerebral endothelial cells. In addition it also provides more precise details of blood brain barrier opening mediated by tissue plasminogen activator

The involvement of dynamical fluctuations in driving enzymatic processes is widely accepted. With respect to NQM tunnelling enzymes, the role of promoting motions in facilitating hydrogenic transfers is well studied. Few studies have however, specifically attributed, dedicated dynamical fluctuations characterised by their timescales and magnitudes, as a function of a reaction coordinate, to specific groups in a protein system. An effectively full suite of backbone resonance assignments were obtained for PETNR and on relevant ligand complexes. This provided an essential platform on which residue specific, backbone amide fluctuations were assessed. This thesis documents the application of pressure up to 1500 bar, in tandem with high resolution TROSY based NMR analysis, as a means of studying residue specific, conformer exchange perturbations. Residue specific amide compression profiles of the PETNR:FMN free enzyme system, and complexes with progesterone and tetrahydropyridine dinucleotides have been obtained. The binding of progesterone appears to induce conformational tightening of residues within the active site vicinity. The complexation of PETNR:FMN with tetrahydropyridine dinucleotides, appears to stimulate conformational shifts towards intermediate, and in some cases, slow exchange regimes in multiple residues about the active site vicinity. This is evidenced by extensive intensity attenuation of 1H-15N TROSY resonances, on the binding of tetrahydropyridine dinucleotides at 1 bar pressure, and on going from 1 bar to 1500 bar pressure. Multiple regions of sequence, spatially clustering about the active site vicinity within a 10 Å sphere of the FMN binding pocket, display appreciable sensitivity to ligand binding. Differential responses of residues to the application of high pressure between complexes was noted within segments of these regions. A region of sequence, named the β-hairpin flap displays significant differential compression profiles between the PETNR:FMN free enzyme system, and associated progesterone and tetrahydropyridine dinucleotide complexes. A role in mediating ligand engagement is proposed for R130 and R142 in the β-hairpin flap. A central hydrogen bonding network, perhaps constituting a putative proton wire in the active site of the PETNR:FMN:Progesterone complex, has been identified that could enable the shuttling of protons following catalytic protonation of oxidative substrate. The resonance response behaviour of G185 acts as a sensitive reporter on the formation of these interactions, revealed by an interrogation of the differences in chemical shift changes on progesterone binding, and in response to high pressure. The recruitment of high resolution crystallographic data sets readily supported a structural and dynamical interpretation of the observed chemical shift responses to ligand binding at 1 bar pressure, and on the application high pressure. A definitive atomistic identification of fast motion contribution to activation barrier compression was not obtained. Nevertheless, detailed, residue specific amide compression profiles, and shifts in backbone amide conformational exchange regimes in response to ground state ligand binding, and at high pressure, have been catalogued in the PETNR:FMN free enzyme system. These dynamical profiles in the free enzyme are contrasted against comparative, residue specific observations in analogue complexes of the oxidative and reductive half reactions of PETNR.

L’injection intraveineuse de l’activateur tissulaire du plasminogène (tPA) est le seul traitement aigu de l’ischémie cérébrale autorisé chez l’Homme. Cependant, la thrombolyse présente des limites d’utilisation, comme son étroite fenêtre thérapeutique, un risque hémorragique et une efficacité de recanalisation malgré tout relativement peu élevée. De plus, la littérature suggère fortement que non seulement le tPA endogène, mais aussi exogène (capable de traverser la barrière hémato-encéphalique), a des effets pro-excitotoxiques. Nous avons proposé que cet effet résulte du clivage de la sous unité NR1 du récepteur NMDA. Malgré un effort important de la communauté scientifique pour chercher de nouveaux traitements, tous les espoirs se sont avérés être des échecs. Sur ces bases, ces travaux de thèse ont consisté à : 1) Améliorer les approches précliniques en développant un nouveau modèle d’ischémie cérébrale chez la souris et en incluant dans les études un des principaux facteur de risque des AVC, le vieillissement ; 2) Développer une stratégie d’immunothérapie visant l’interaction tPA/ récepteur NMDA. J’ai ainsi montré qu’il existe une diminution du volume de lésion ischémique corrélée à l’âge et que cette diminution de tPA est due à une diminution d’expression du facteur de transcription D-Site Albumin Binding Protein (DBP). J’ai également développé un modèle innovant d’ischémie thrombo-embolique chez la souris, dans lequel la reperfusion par le tPA est bénéfique, si tant est qu’elle soit réalisée de manière précoce. Sur ce modèle, j’ai apporté par une stratégie d’immunisation active la preuve in vivo du clivage du domaine amino-terminal de la sous-unité NR1 des récepteurs NMDA. Enfin, j’ai produit un anticorps médicament, capable d’empêcher l’interaction du tPA avec la sous-unité NR1 des récepteurs NMDA, dont une injection unique permet de réduire les lésions ischémiques, mais aussi d’augmenter la fenêtre thérapeutique de la thrombolyse, conférant alors une récupération fonctionnelle à long terme. Cette stratégie pourrait donc accroître la proportion de patients traitables après un AVC ischémique aiguë
Reperfusion with tissue plasminogen activator (tPA) is the only approved treatment for ischemic stroke. However, thrombolysis has some limitations, including a narrow therapeutic window, an elevated risk of hemorrhage transformation and a low level of effective recanalization. Moreover, there is a growing body of evidence that both endogenous and exogenous tPA (able to cross the blood-brain barrier) could mediated pro-excitotoxic effects. We have proposed that this noxious effect results from the cleavage of the NR1 subunit of the NMDA receptor. My thesis work consisted in: 1) Improving pre-clinic approaches by developing a new model of thrombo-embolic ischemia in mice and by taking into account a major risk factor for stroke, aging; 2) Developing a strategy of immunotherapy targeting the interaction between tPA and NMDA receptor. I have thus shown that ischemic lesions decrease as a function of age, due to reduced levels of tPA. Moreover, I have identified DBP (D-site albumin Binding Protein), as being the transcription factor responsible for the control of tPA levels as a function of age. I have also developed a new model of thrombo-embolic ischemia in mice, in which tPA-induced thrombolysis is beneficial, provided it is performed soon enough. In this model, I have demonstrated by using a strategy of active immunization the in vivo occurrence of the cleavage of the NMDA receptor NR1 subunit by tPA. Finally, I have produced an antibody able to prevent the interaction between tPA and the NMDA receptor subunit, of which a single injection confers long lasting brain protection and neurological recovery and can also increase the therapeutic window of thrombolysis. This strategy could thus significantly increase the proportion of treatable ischemic stroke patients

Le paf (1-o-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) est aujourd'hui reconnu comme un des plus importants neuromediateurs lipidiques. Dans ce travail, nous montrons une action du paf sur les cellules de la lignee de neuroblastome n1e-115. Par les techniques de liaison, nous avons mis en evidence sur ces cellules la presence de deux types de recepteurs du paf. L'activation de ces recepteurs par des concentrations physiologiques de paf produit une accumulation d'inositol triphosphate, et une augmentation rapide et transitoire du calcium libre cytosolique. Cette augmentation du calcium libre cytosolique fait intervenir a la fois une redistribution du calcium intracellulaire et des influx de calcium externe. Ces influx de calcium passent essentiellement via des canaux permeables aux ions ca#2#+ de type receptor-operated channel. Nous montrons la presence, sur ces cellules, de canaux permeables au ca#2#+ et activables par la thapsigargine correspondant tres probablement a des canaux calciques activables via la depletion des reserves intracellulaires. Ces canaux interviennent dans la reponse induite par le paf. A l'aide de la technique de voltage impose sur cellule entiere, nous avons mis en evidence une activation possible par le paf des canaux calciques sensibles au voltage de type l. Cependant, cette derniere action du paf ne participe que, de facon minime, aux entrees de ca#2#+. L'activation de ces canaux est, en effet, bloquee par une hyperpolarisation transitoire en reponse a l'activation par le paf de canaux potassiques du type bk(ca). Cette derniere action du paf est la consequence de la seule augmentation du calcium libre cytosolique, et ne resulte pas d'une action directe du paf sur ces bk(ca). Enfin, a l'aide de la technique du patch-clamp, nous n'avons pas mis en evidence l'effet du paf sur les autres permeabilites ioniques membranaires de ces cellules (courants na#+ et k#+ sensibles au potentiel)

Les accidents vasculaires cérébraux (AVC) constituent un problème majeur de santé publique. Ils sont en majorité de type ischémique, c’est-à-dire liés à l’occlusion d’une artère cérébrale. Le seul traitement actuel de ces AVC ischémiques est la thrombolyse par l’activateur tissulaire du plasminogène recombinant (rt-PA). Cependant, ce traitement est associé à un risque élevé d’hémorragies intracérébrales post-ischémiques, encore appelées transformations hémorragiques (TH), qui contribuent à la dégradation neurologique des patients. Il apparaît donc indispensable de développer des stratégies à associer au rt-PA, afin de protéger le lit vasculaire et de réduire les TH. L’objectif de ce travail de thèse était d’étudier l’implication d’une enzyme, la poly(ADP-ribose)polymérase ou PARP, dans les effets délétères du rt-PA, et plus particulièrement au niveau de la barrière hémato-encéphalique (BHE). Nos travaux ont été menés dans un modèle d’ischémie cérébrale réalisé chez la souris. Dans ce modèle, nous avons mis en évidence le rôle de la PARP dans les TH induites par le rt-PA, grâce à deux techniques : le Western blot d’hémoglobine, permettant d’évaluer la quantité de sang présente dans le parenchyme cérébral, et l’Imagerie par Résonnance Magnétique. Afin de préciser les cibles de la PARP sous-tendant sa contribution aux TH post-thrombolyse, nous nous sommes intéressés à différents constituants de la BHE : la claudine-5, l’occludine et ZO-1 (zonula occludens-1), protéines des jonctions serrées, la VE-cadhérine des jonctions adhérentes et le collagène IV et la laminine, constituants de la lame basale. Nous avons montré que l’ischémie s’accompagne d’une dégradation de la claudine-5, de ZO-1, et de la VE-cadhérine qui est aggravée par le rt-PA ; l’administration d’un puissant inhibiteur de PARP, le PJ34, permet de s’opposer à la dégradation de ces protéines par le rt-PA. Une réduction de la dégradation de la laminine par le rt-PA a également été observée avec le PJ34. Grâce à une collaboration avec le Pr Bérézowski de Lens, nous avons pu montrer dans un modèle in vitro que le PJ34 est capable de traverser la BHE, à la fois dans des conditions « physiologiques » et dans des conditions mimant l’ischémie cérébrale (oxygen/glucose deprivation). Afin de déterminer les voies de signalisation modulées par la PARP conduisant à la dégradation de la BHE et aux TH, nous avons travaillé sur un modèle in vitro de cultures de cellules endothéliales (lignée bEnd.3). Sur ce modèle, nous avons d’ores et déjà pu mettre en évidence une mort cellulaire après un stress excitotoxique et le rôle de la PARP dans cette mort. L’ensemble de ces travaux a permis de démontrer le rôle de la PARP dans la dégradation de différents constituants de la BHE par le rt-PA à la suite de l’ischémie cérébrale. Les futures études in vitro sur cultures cellulaires devraient nous permettre d’explorer les mécanismes mis en jeu dans cette situation pathologique. Une meilleure connaissance de ces mécanismes renforcera l’intérêt des inhibiteurs de PARP pour la prévention des TH post-thrombolyse chez les patients victimes d’AVC ischémiques
Stroke is a leading public health problem, the majority of which is ischemic, i.e. caused by the occlusion of a cerebral artery. The only pharmacological approved treatment for acute ischemic stroke is thrombolysis by recombinant tissue plasminogen activator (rt-PA). However, this treatment increases the risk of intracerebral hemorrhages, also called hemorrhagic transformations (HT), which contribute to the neurologic aggravation of the patients. It therefore appears essential to develop strategies protecting the vascular bed after cerebral ischemia in order to reduce these HT. The aim of the present work was therefore to study the implication of a nuclear enzyme, the poly(ADP-ribose)polymerase (PARP) in the vascular effects of rt-PA , with special concern for the blood-brain barrier (BBB). Focal cerebral ischemia was performed in mice by permanent endovascular occlusion of the left middle cerebral artery. In this model, we demonstrated the role of PARP in the rt-PA induced HT by two methods: the Western blot of hemoglobin to evaluate the quantity of blood in the cerebral parenchyma, and magnetic resonance imaging. In order to clarify the targets of PARP underlying its contribution to post-thrombolysis HT, we studied several components of the BBB by Western blot: proteins of tight junctions [claudin-5, occludin and zonula occludens-1 (ZO-1)], protein of adherens junction (VE-cadherin) and proteins of basal membrane (collagen IV and laminin). We demonstrated that ischemia induced a marked decrease of claudin-5, ZO-1 and VE-cadherin, which was aggravated by rt-PA. Administration of a potent PARP inhibitor, PJ34, counteracted the degradation of these proteins by rt-PA. A reduction of the degradation of the laminin by rt-PA was also shown with PJ34. Thanks to a collaboration with Pr Berezowski from Lens, we showed in an in vitro BBB model that PJ34 is able to cross the BBB in physiological condition and during oxygen and glucose deprivation, a condition that mimicks cerebral ischemia. In order to determine the molecular pathways modulated by PARP leading to the degradation of the BBB and to HT, we developed an in vitro model of endothelial cell culture (cell line bEnd.3). In this model, we have already shown a cell death after an excitotoxic stress and the role of PARP in this cell death. This work thus demonstrated the role of PARP in the degradation of different components of the BBB induced by rt-PA after cerebral ischemia. The future in vitro studies on cell culture will enable us to further understand the mechanisms implicated in this pathologic situation. A better knowledge of these mechanisms will increase the interest of the use of PARP inhibitors in the prevention of post-thrombolysis HT in patients suffering from ischemic stroke

The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. Helicobacter pylori might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of H. pylori on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat.

A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (<20μm) and discontinuous in the small intestine.

Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from H. pylori (HPE). In animals with a chronic H. pylori infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum.

HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.

Organic semiconductors are a new key technology for large-area and flexible thin-film electronics. They are deposited as thin films (sub-nanometer to micrometer) on large-area substrates. The technologically most advanced applications are organic light emitting diodes (OLEDs) and organic photovoltaics (OPV). For the improvement of performance and efficiency, correct modeling of the electronic processes in the devices is essential. Reliable characterization and validation of the electronic properties of the materials is simultaneously required for the successful optimization of devices. Furthermore, understanding the relations between material structures and their key characteristics opens the path for innovative material and device design. In this thesis, two material characterization methods are developed, respectively refined and applied: a novel technique for measuring the charge carrier mobility μ and a way to determine the ionization energy IE or the electron affinity EA of an organic semiconductor. For the mobility measurements, a new evaluation approach for space-charge limited current (SCLC) measurements in single carrier devices is developed. It is based on a layer thickness variation of the material under investigation. In the \"potential mapping\" (POEM) approach, the voltage as a function of the device thickness V(d) at a given current density is shown to coincide with the spatial distribution of the electric potential V(x) in the thickest device. On this basis, the mobility is directly obtained as function of the electric field F and the charge carrier density n. The evaluation is model-free, i.e. a model for μ(F, n) to fit the measurement data is not required, and the measurement is independent of a possible injection barrier or potential drop at non-optimal contacts. The obtained μ(F, n) function describes the effective average mobility of free and trapped charge carriers. This approach realistically describes charge transport in energetically disordered materials, where a clear differentiation between trapped and free charges is impossible or arbitrary. The measurement of IE and EA is performed by characterizing solar cells at varying temperature T. In suitably designed devices based on a bulk heterojunction (BHJ), the open-circuit voltage Voc is a linear function of T with negative slope in the whole measured range down to 180K. The extrapolation to temperature zero V0 = Voc(T → 0K) is confirmed to equal the effective gap Egeff, i.e. the difference between the EA of the acceptor and the IE of the donor. The successive variation of different components of the devices and testing their influence on V0 verifies the relation V0 = Egeff. On this basis, the IE or EA of a material can be determined in a BHJ with a material where the complementary value is known. The measurement is applied to a number of material combinations, confirming, refining, and complementing previously reported values from ultraviolet photo electron spectroscopy (UPS) and inverse photo electron spectroscopy (IPES). These measurements are applied to small molecule organic semiconductors, including mixed layers. In blends of zinc-phthalocyanine (ZnPc) and C60, the hole mobility is found to be thermally and field activated, as well as increasing with charge density. Varying the mixing ratio, the hole mobility is found to increase with increasing ZnPc content, while the effective gap stays unchanged. A number of further materials and material blends are characterized with respect to hole and electron mobility and the effective gap, including highly diluted donor blends, which have been little investigated before. In all materials, a pronounced field activation of the mobility is observed. The results enable an improved detailed description of the working principle of organic solar cells and support the future design of highly efficient and optimized devices
Organische Halbleiter sind eine neue Schlüsseltechnologie für großflächige und flexible Dünnschichtelektronik. Sie werden als dünne Materialschichten (Sub-Nanometer bis Mikrometer) auf großflächige Substrate aufgebracht. Die technologisch am weitesten fortgeschrittenen Anwendungen sind organische Leuchtdioden (OLEDs) und organische Photovoltaik (OPV). Zur weiteren Steigerung von Leistungsfähigkeit und Effizienz ist die genaue Modellierung elektronischer Prozesse in den Bauteilen von grundlegender Bedeutung. Für die erfolgreiche Optimierung von Bauteilen ist eine zuverlässige Charakterisierung und Validierung der elektronischen Materialeigenschaften gleichermaßen erforderlich. Außerdem eröffnet das Verständnis der Zusammenhänge zwischen Materialstruktur und -eigenschaften einen Weg für innovative Material- und Bauteilentwicklung. Im Rahmen dieser Dissertation werden zwei Methoden für die Materialcharakterisierung entwickelt, verfeinert und angewandt: eine neuartige Methode zur Messung der Ladungsträgerbeweglichkeit μ und eine Möglichkeit zur Bestimmung der Ionisierungsenergie IE oder der Elektronenaffinität EA eines organischen Halbleiters. Für die Beweglichkeitsmessungen wird eine neue Auswertungsmethode für raumladungsbegrenzte Ströme (SCLC) in unipolaren Bauteilen entwickelt. Sie basiert auf einer Schichtdickenvariation des zu charakterisierenden Materials. In einem Ansatz zur räumlichen Abbildung des elektrischen Potentials (\"potential mapping\", POEM) wird gezeigt, dass das elektrische Potential als Funktion der Schichtdicke V(d) bei einer gegebenen Stromdichte dem räumlichen Verlauf des elektrischen Potentials V(x) im dicksten Bauteil entspricht. Daraus kann die Beweglichkeit als Funktion des elektrischen Felds F und der Ladungsträgerdichte n berechnet werden. Die Auswertung ist modellfrei, d.h. ein Modell zum Angleichen der Messdaten ist für die Berechnung von μ(F, n) nicht erforderlich. Die Messung ist außerdem unabhängig von einer möglichen Injektionsbarriere oder einer Potentialstufe an nicht-idealen Kontakten. Die gemessene Funktion μ(F, n) beschreibt die effektive durchschnittliche Beweglichkeit aller freien und in Fallenzuständen gefangenen Ladungsträger. Dieser Zugang beschreibt den Ladungstransport in energetisch ungeordneten Materialien realistisch, wo eine klare Unterscheidung zwischen freien und Fallenzuständen nicht möglich oder willkürlich ist. Die Messung von IE und EA wird mithilfe temperaturabhängiger Messungen an Solarzellen durchgeführt. In geeigneten Bauteilen mit einem Mischschicht-Heteroübergang (\"bulk heterojunction\" BHJ) ist die Leerlaufspannung Voc im gesamten Messbereich oberhalb 180K eine linear fallende Funktion der Temperatur T. Es kann bestätigt werden, dass die Extrapolation zum Temperaturnullpunkt V0 = Voc(T → 0K) mit der effektiven Energielücke Egeff , d.h. der Differenz zwischen EA des Akzeptor-Materials und IE des Donator-Materials, übereinstimmt. Die systematische schrittweise Variation einzelner Bestandteile der Solarzellen und die Überprüfung des Einflusses auf V0 bestätigen die Beziehung V0 = Egeff. Damit kann die IE oder EA eines Materials bestimmt werden, indem man es in einem BHJ mit einem Material kombiniert, dessen komplementärer Wert bekannt ist. Messungen per Ultraviolett-Photoelektronenspektroskopie (UPS) und inverser Photoelektronenspektroskopie (IPES) werden damit bestätigt, präzisiert und ergänzt. Die beiden entwickelten Messmethoden werden auf organische Halbleiter aus kleinen Molekülen einschließlich Mischschichten angewandt. In Mischschichten aus Zink-Phthalocyanin (ZnPc) und C60 wird eine Löcherbeweglichkeit gemessen, die sowohl thermisch als auch feld- und ladungsträgerdichteaktiviert ist. Wenn das Mischverhältnis variiert wird, steigt die Löcherbeweglichkeit mit zunehmendem ZnPc-Anteil, während die effektive Energielücke unverändert bleibt. Verschiedene weitere Materialien und Materialmischungen werden hinsichtlich Löcher- und Elektronenbeweglichkeit sowie ihrer Energielücke charakterisiert, einschließlich bisher wenig untersuchter hochverdünnter Donator-Systeme. In allen Materialien wird eine deutliche Feldaktivierung der Beweglichkeit beobachtet. Die Ergebnisse ermöglichen eine verbesserte Beschreibung der detaillierten Funktionsweise organischer Solarzellen und unterstützen die künftige Entwicklung hocheffizienter und optimierter Bauteile

The main objective of this thesis is a comprehensive assessment of the practical usability and effectiveness of modern activating teaching methods in economic subjects in fulfilling the RVP economics and business and RVP Business Academy in comparison with traditional (standard) methods. To achieve this goal, a systematic clarification and evaluation of key elements of the choice of adequate methods of teaching, presentation and comparison of traditional, modern activating and comprehensive teaching methods was employed. Selected modern activating methods are described in detail, including the procedure and examples of their application in the teaching of management on the circuit of the secondary vocational schools. Current status in the use of teaching methods by teachers of economic subjects on selected secondary vocational schools was mapped and assessed through quantitative (structured interviewing of students and teachers) and qualitative (interview with trachet of economic subjects at selected schools, testing of students -- pretest and posttest) methods of empirical educational research and realization of experimental teaching. The results of the analysis of the usefulness and effectiveness of modern activating methods in achieving the educational goals of economic education in secondary vocational schools are summarized in the form of a SWOT analysis. The work, inter alia, examines and assesses the barriers in the application of modern methods of teaching, to what extent these methods contribute to the building of the desired competences of students and in particular the so-called, soft skills of students, verifies the effectiveness of the textbooks Management based on modern activating methods in meeting educational goals. The thesis provides outputs for management of secondary schools including, in particular, the RVP Business Academy and RVP economics and business, which may become a guideline, the stimulus for change methods and forms of teaching, the need to offset the interest of schools from traditional methods to modern activating methods. It can become a tool and an inspiration for each of economic subjects in the adoption and subsequent implementation of modern teaching methods, since it contains a procedure for their application. The intention of the processing of the research problem and solved in the framework of this thesis was to contribute to pedagogical theory and practice the findings supporting the need for qualitative changes in the process. It offers practical recommendations and observations, how should a teacher of economic subjects at secondary vocational schools to proceed with the introduction of modern activating methods. The chosen research methods and processes to provide a relatively large amount of data, the processing to get a deeper insight on the role, effectiveness and applicability of modern teaching methods in the environment of the contemporary secondary economic education.

Le seul traitement actuellement disponible pour les accidents vasculaires cérébraux d’origine ischémique est la thrombolyse par l’activateur tissulaire du plasminogène recombinant (rt-PA). Cependant, l’efficacité du rt-PA est souvent partielle ou absente, et des phénomènes de réocclusion du vaisseau peuvent être observés. Par ailleurs, l’administration de rt-PA est associée à un risque hémorragique. Il apparaît donc indispensable de rechercher les mécanismes à l’origine de la toxicité vasculaire du rt-PA, afin de pouvoir développer des stratégies capables de protéger le lit vasculaire. Parmi ces stratégies, notre équipe a montré dans des modèles expérimentaux que l’inhibition d’une enzyme nucléaire, la poly(ADP-ribose) polymérase ou PARP, permet de protéger la barrière hémato-encéphalique, de réduire les hémorragies et d’améliorer la reperfusion cérébrale suite à l’administration post-ischémique de rt-PA. Dans ce contexte, mon travail a consisté à étudier les mécanismes impliqués dans les altérations vasculaires associées à l’administration de rt-PA à la suite de l’ischémie. Mes travaux de recherche ont comporté un volet in vivo et un volet in vitro. Les études réalisées in vivo ont été menées dans un modèle murin d’ischémie cérébrale thrombo-embolique. Nos résultats indiquent que ni l’ischémie, ni le rt-PA, ni l’association au rt-PA d’un puissant inhibiteur de PARP, le PJ34, ne modifient à 24 heures la présence de dépôts de fibrine, marqueur d’hypoperfusion et de réocclusion. Nous nous sommes ensuite intéressés à deux marqueurs endothéliaux d’inflammation : VCAM-1 et ICAM-1, et avons montré que leur expression, qui augmente 24 heures après l’ischémie, n’est pas modifiée par le rt-PA. Enfin, l’association du PJ34 au rt-PA réduit significativement l’expression post-ischémique de VCAM-1, ce qui suggère le rôle de la PARP dans l’expression de cette molécule d’adhésion. La seconde partie de mon travail a été réalisée in vitro sur une lignée de cellules endothéliales cérébrales murines (bEnd.3). Le rt-PA est à l’origine de changements caractéristiques au niveau de l’organisation et de la morphologie de ces cellules. Ces changements ne sont pourtant associés ni à une dégradation de l’expression des molécules de jonctions inter-endothéliales (occludine, VE-cadhérine), ni à une augmentation de l’expression des marqueurs endothéliaux pro-inflammatoires (VCAM-1, ICAM-1). Nous nous sommes également intéressés à d’autres marqueurs de dysfonction endothéliale, les microparticules endothéliales (MPE). Nos résultats montrent que le rt-PA est à l’origine d’une augmentation importante de la libération des MPE. L’utilisation d’un inhibiteur de la protéine p38, le SB203580, et d’un inhibiteur de PARP, le PJ34, permet de réduire cette augmentation, ce qui suggère que p38 et la PARP pourraient être impliquées dans la production de MPE induite par le rt-PA. En conclusion, l’ensemble de ce travail contribue à préciser les effets vasculaires du rt-PA. Parmi ces effets, la mise en évidence de la production de MPE, via la PARP, est particulièrement novatrice
Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, the efficacy of rt-PA is rarely complete, and arterial reocclusion can be observed. Furthermore, administration of rt-PA increases the risk of hemorrhagic transformations. Therefore, it is essential to seek mechanisms underlying the vascular toxicity of rt-PA in order to develop strategies protecting the vascular bed. Among these strategies, our laboratory has previously shown that inhibition of poly (ADP-ribose) polymerase (PARP), a nuclear enzyme, protects the blood-brain barrier, reduces hemorrhagic transformations and improves cerebral reperfusion following the post-ischemic administration of rt-PA. In this context, the aim of the present work was to establish the post-ischemic mechanisms of rt-PA-induced vascular alterations. The research was divided into (1) in vivo experiments and (2) in vitro studies to examine the effect of rt-PA on the endothelium. The in vivo studies were performed in a mouse model of thrombo-embolic stroke induced by thrombin injection in the middle cerebral artery. Our results showed that neither ischemia, nor rt-PA, nor the association to rt-PA of the potent inhibitor of PARP PJ34 alter cerebral fibrin deposits, a marker of hypoperfusion and reocclusion, at 24 hours after ischemia. We then evaluated the expression of two endothelial markers of inflammation : VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1). Our results showed that their expressions increase 24 hours after ischemia and are not modified by rt-PA. Finally, the association of PJ34 to rt-PA significantly reduced the post-ischemic expression of VCAM-1, suggesting a role for PARP in the expression of this adhesion molecule. The second part of my work was carried out in vitro in cultures of mouse brain-derived endothelial cells bEnd.3. In the presence of rt-PA, the organization and the morphology of the endothelial cells radically changed. However, these changes were associated neither to a degradation of endothelial junction proteins (occludin, VE-cadherin (vascular endothelial-cadherin)), nor to an increase in the expression of pro-inflammatory endothelial markers (VCAM-1, ICAM-1). We were also interested in a recently identified marker of endothelial dysfunction : endothelial microparticles (EMP). Our results showed that rt-PA induces a significant increase in the EMP released by bEnd.3 cells. The use of a p38 inhibitor, SB203580, and the PARP inhibitor, PJ34, reduced this increase, suggesting that p38 and PARP could be involved in the EMP production induced by rt-PA. In conclusion, this work helps to clarify the vascular effects of rt-PA. Among these effects, the highlight of EMP production, through PARP pathway, is particularly original

碩士
元智大學
化學工程與材料科學學系
107
In the research, we have conducted the linear dielectric barrier discharge (DBD) atmospheric pressure plasma for surface modification on Polytetrafluoroethylene (PTFE) surface by plasma inducing acrylic acid (AAc) grafting polymerization. An overarching goal of our research is to increase hydrophilicity on PTFE surface after plasma treatment. First, DBD plasma is used to activate PTFE substrate and determine the activation effect with four operational conditions. Second, acrylic acid (AAc) is induced grafting polymerization on PTFE substrate. It is elucidated that the different grafting results with three grafting methods. The first method is that the PTFE was immersed in the AAc solution with the DBD plasma pre-treatment. The second method is that the PTFE was treated by DBD plasma after immersing in AAc solution. The third method is using DBD plasma to activate the PTFE in the beginning. Then, the DBD plasma treated PTFE again after the activated PTFE immersed in the AAc solution. In addition, the optimized third grafting method is found to apply for different polymers and compare the degree of grafting. The contact angle meter was used to detect the wettability of the surface. And the surface free energy, polar component and dispersive component are calculated by Owens-Wendt-Rabel-Kaelble (OWRK). FTIR-ATR and XPS were used to detect the change of the surface functional groups and surface element contents of the DBD plasma treated PTFE. The fitting of C1s peaks can be used to derive the possible functional groups of treated PTFE substrate. SEM detected the surface morphology analysis. The degree of grafting was also examined in this research. ESR was employed to detect the intensity of the free radicals on the treated PTFE. It was found that the surface free energy of PTFE can be enhanced by DBD plasma. In the AAc grafting part, the third grafting method was found to obtain the higher degree of grafting. The high monomer concentration for AAc grafting polymerization can obtain superior wettability. The research represents that Linear DBD plasma is an effective technique on surface modification for fluorocarbon polymer.

Yes
Background and Objective Visible light has beneficial effects on cutaneous wound healing, but the role of potential photoreceptors in human skin is unknown. In addition, inconsistency in the parameters of blue and red light‐based therapies for skin conditions makes interpretation difficult. Red light can activate cytochrome c oxidase and has been proposed as a wound healing therapy. UV‐blue light can activate Opsin 1‐SW, Opsin 2, Opsin 3, Opsin 4, and Opsin 5 receptors, triggering biological responses, but their role in human skin physiology is unclear. Materials and Methods Localization of Opsins was analyzed in situ in human skin derived from face and abdomen by immunohistochemistry. An ex vivo human skin wound healing model was established and expression of Opsins confirmed by immunohistochemistry. The rate of wound closure was quantitated after irradiation with blue and red light and mRNA was extracted from the regenerating epithelial tongue by laser micro‐dissection to detect changes in Opsin 3 (OPN3) expression. Retention of the expression of Opsins in primary cultures of human epidermal keratinocytes and dermal fibroblasts was confirmed by qRT‐PCR and immunocytochemistry. Modulation of metabolic activity by visible light was studied. Furthermore, migration in a scratch‐wound assay, DNA synthesis and differentiation of epidermal keratinocytes was established following irradiation with blue light. A role for OPN3 in keratinocytes was investigated by gene silencing. Results Opsin receptors (OPN1‐SW, 3 and 5) were similarly localized in the epidermis of human facial and abdominal skin in situ. Corresponding expression was confirmed in the regenerating epithelial tongue of ex vivo wounds after 2 days in culture, and irradiation with blue light stimulated wound closure, with a corresponding increase in OPN3 expression. Expression of Opsins was retained in primary cultures of epidermal keratinocytes and dermal fibroblasts. Both blue and red light stimulated the metabolic activity of cultured keratinocytes. Low levels of blue light reduced DNA synthesis and stimulated differentiation of keratinocytes. While low levels of blue light did not alter keratinocyte migration in a scratch wound assay, higher levels inhibited migration. Gene silencing of OPN3 in keratinocytes was effective (87% reduction). The rate of DNA synthesis in OPN3 knockdown keratinocytes did not change following irradiation with blue light, however, the level of differentiation was decreased. Conclusions Opsins are expressed in the epidermis and dermis of human skin and in the newly regenerating epidermis following wounding. An increase in OPN3 expression in the epithelial tongue may be a potential mechanism for the stimulation of wound closure by blue light. Since keratinocytes and fibroblasts retain their expression of Opsins in culture, they provide a good model to investigate the mechanism of blue light in wound healing responses. Knockdown of OPN3 led to a reduction in early differentiation of keratinocytes following irradiation with blue light, suggesting OPN3 is required for restoration of the barrier function. Understanding the function and relationship of different photoreceptors and their response to specific light parameters will lead to the development of reliable light‐based therapies for cutaneous wound healing.
European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no.: 607886

Tese de mestrado, Ciências Biofarmacêuticas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014
The blood-brain barrier (BBB) is more than a loyal protecting wall of the central nervous system (CNS). The BBB is a dynamic bidirectional interface between the CNS and blood, formed by endothelial cells, basement membrane, pericytes and astrocytes endfeet. Since its unique properties and location, it is a central player in the maintenance of CNS microenvironment. The communication between the BBB and the neurovascular unit components, microglia and neurons, was found to be crucial for the CNS homeostasis, as it was found to be dysfunctional in aged brain and in Alzheimer’s disease (AD) patients brain. Based on this, we aimed to investigate which vascular and glial events are characteristic of AD or/and aging, as well as to establish the temporal evolution of these changes in AD-like APP/PS1 and wild-type (WT) mice. Moreover, we aimed to relate these changes with amyloid-β (Aβ) accumulation. We used hippocampi and cortex to analyze the temporal evolution of selected parameters in a young adult, a middle age and an old age group. Our results show that aging is the main factor contributing to the upregulation of receptor for advanced glycation endproducts and desmin, as well as to the entrance of thrombin and albumin in hippocampus parenchyma. On the other hand, AD was found to be the unique contributing factor to the loss of platelet-derived growth factor receptor-β (PDGFR-β) positive cells, in both studied regions. Both factors contributed to hypovascularization in hippocampus, but in cortex it was just a reflex of the interaction between both factors. Astrogliosis was a result of AD in hippocampus and it is a reflex of both factors in cortex, while microgliosis is a result of AD and the interaction between both factors in both regions. Regarding the relationship between glia-vascular changes and senile plaques, we found that senile plaques precede vascular and glial alterations in hippocampus. Interestingly, in cortex, vascular and glial alterations, specifically loss of PDGFR-β-positive cells and astrogliosis, accompanied the first senile plaques. In sum, this study points to vascular and glial events that can underline AD pathogenesis and age-related brain vulnerabilities.
A barreira hematoencefálica (BHE) é mais do que uma simples barreira protetora do sistema nervoso central (SNC). A BHE é uma barreira dinâmica e bidirecional entre o SNC e o sangue, formado por células endoteliais, membrana basal, pericitos e as terminações dos astrócitos. O facto de a BHE estar localizada numa posição privilegiada e de ter propriedades únicas, permite-a desempenhar funções de manutenção na homeostasia do SNC. A perturbação da comunicação entre a BHE e os elementos da unidade neurovascular, a microglia e os neurónios, parece ser uma característica do envelhecimento e da doença de Alzheimer (DA). Deste modo, o objetivo deste trabalho foi investigar se as alterações vasculares e gliais são características do envelhecimento e/ou da DA e estabelecer a evolução temporal dessas alterações ao longo do envelhecimento, em ratinhos saudáveis (wild-type), e da progressão da doença, utilizando o modelo APP/PS1 que mimetiza a DA. Além disso, essas alterações foram relacionadas com a densidade das placas senis. Foram utilizados o hipocampo e o córtex de três grupos diferentes, um grupo de jovens adultos, um grupo de indivíduos de meia-idade e um terceiro grupo constituído por indivíduos idosos, de modo a analisar a evolução temporal dos parâmetros selecionados. Os resultados obtidos demonstram que o envelhecimento é o principal fator que contribui para o aumento da expressão do recetor dos produtos avançados da glicação e de desmina, bem como para a entrada de trombina e albumina para o parênquima do hipocampo. Por outro lado, a perda de células positivas para o recetor do fator de crescimento derivado de plaquetas (PDGFR-β) em ambas as regiões foi o resultado da DA. Ambos os fatores estudados contribuíram para a hipovascularização no hipocampo, mas no córtex foi um resultado da interação entre ambos os fatores. A astrogliose é o resultado da DA no hipocampo, enquanto que no córtex isso é o resultado de ambos os fatores. A microgliose é afetada pela DA e pela interação entre ambos os fatores em ambas as regiões. Considerando a relação entre as alterações gliais e vasculares com o aparecimento de placas senis, foi estabelecido que as placas senis precedem as mudanças gliais e vasculares apenas no hipocampo. Interessantemente, no córtex as relações gliais e vasculares, nomeadamente a perda de células positivas para o PDGFR-β e a astrogliose, são acompanhadas pelo aparecimento de placas senis. Deste modo, este estudo aponta para o facto de as alterações vasculares e gliais podem estar associadas à patogénese da DA e à vulnerabilidade do cérebro a patologias associadas à idade.
Fundação para a Ciência e a Tecnologia e fundos filantrópicos

A study of defects in semiconductors is vital as defects tend to influence device operation by modifying their electrical and optoelectronic properties. This influence can at times be desirable in the case of fast switching devices and sometimes undesirable as they may reduce the efficiency of optoelectronic devices. ZnO is a wide bandgap material with a potential for fabricating UV light emitting diodes, lasers and white lighting devices only after the realization of reproducible p-type material. The realization of p-type material is greatly affected by doping asymmetry. The self-compensation behaviour by its native defects has hindered the success in obtaining the p-type material. Hence there is need to understand the electronic properties, formation and annealing-out of these defects for controlled material doping. Space charge spectroscopic techniques are powerful tools for studying the electronic properties of electrically active defects in semiconductors since they can reveal information about the defect “signatures”. In this study, novel Schottky contacts with low leakage currents of the order of 10-11 A at 2.0 V, barrier heights of 0.60 – 0.80 eV and low series resistance, fabricated on hydrogen peroxide treated melt-grown single crystal ZnO samples, were demonstrated. Investigations on the dependence of the Schottky contact parameters on fabrication techniques and different metals were performed. Resistive evaporation proved to produce contacts with lower series resistance, higher barrier heights and low reverse currents compared to the electron-beam deposition technique. Deep level transient spectroscopy (DLTS) and Laplace-DLTS have been employed to study the electronic properties of electrically active deep level defects in ZnO. Results revealed the presence of three prominent deep level defects (E1, E2 and E3) in the as-received ZnO samples. Electron-beam deposited contacts indicated the presence of the E1, E2 and E3 and the introduction of new deep level defects. These induced deep levels have been attributed to stray electrons and ionized particles, present in the deposition system during contact fabrication. Exposure of ZnO to high temperatures induces deep level defects. Annealing samples in the 300°C – 600°C temperature range in Ar + O2 induces the E4 deep level with a very high capture cross-section. This deep level transforms at every annealing temperature. Its instability at room temperature has been demonstrated by a change in the peak temperature position with time. This deep level was broad, indicating that it consists of two or more closely spaced energy levels. Laplace-DLTS was successfully employed to resolve the closely spaced energy levels. Annealing samples at 700°C in Ar and O2 anneals-out E4 and induces the Ex deep level defect with an activation enthalpy of approximately 160 – 180 meV. Vacuum annealing performed in the 400°C – 700°C temperature range did not induce any deep level defects. Since the radiation hardness of ZnO is crucial in space applications, 1.6 MeV proton irradiation was performed. DLTS revealed the introduction of the E4 deep level with an activation enthalpy of approximately 530 meV, which proved to be stable at room temperature and atmospheric pressure since its properties didn’t change over a period of 12 months.
Thesis (PhD)--University of Pretoria, 2013.
Physics
unrestricted

Ce travail de thèse porte sur l'étude du décalage d'échange dans le system Co / MnPd.
Les échantillons ont été caractérisée structuralement en utilisant la diffraction et la réflectométrie de rayons X et magnétiquement en utilisant un magnétomètre VSM et un magnétomètre SQUID pour les mesures magnétiques.
Compte tenu de la difficulté dans la compréhension des phénomènes physiques impliqués dans le décalage d'échange, un modèle phénoménologique simple a été développé dans lequel le champ magnétique moléculaire sur la couche antiferromagnétique est modélisé comme un champ magnétique extérieur appliqué. La valeur du champ de décalage est déterminée par le couplage existant entre la première et la deuxième couche AFM.
L'étude des effets traînage dans les systèmes à décalage d'échange, constitue une originalité de ce travail. Le modèle de Fatuzzo Labrune, très souvent utilisé pour décrire le renversement de l'aimantation dans les couches magnétiques dures a permis la description de l'activation thermique sur la première partie du cycle dé hysteresis. Ce modèle est valable lorsqu'on considère une seule barrière d'énergie dans la nucléation des domaines magnétiques et une seule barrière pour la propagation des parois. Au delà du premier cycle d'hysteresis, les résultat expérimentaux ne peuvent être décrits par ce modèle. Les modèles qui supposent l'existence d'une large distribution des barrières d'énergie permet alors une très bonne description des effets observés.

The Diels-Alder reaction was discovered in 1928 and has become the most efficient and practical method for the synthesis of six-membered carbocyclic and heterocyclic rings. This thesis comprises three chapters of results and discussion with the Diels-Alder reaction as a theme. Chapter 2 details an investigation of endo:exo selectivity in the Diels-Alder reactions of 1,3-butadiene. Chapter 3 explores aspects of the intramolecular Diels-Alder reactions of some substituted 1,3,8-nonatrienes, and Chapter 4 describes the domino Diels-Alder reactions of 1,4-diiodo-1,3-butadiene. The Diels-Alder reaction is powerful, general, and widely used in chemical synthesis, and it is well known that many Diels-Alder reactions exhibit endo selectivity, in accord with Alder’s empirical rule. The origins of endo:exo selectivity in the Diels-Alder reaction, however, are not completely understood and there is a dearth of experimental evidence concerning the Diels-Alder reactions of the archetypal 1,3-diene, 1,3- butadiene. Chapter 2 describes a study of the Diels-Alder reactions of an isotopically labelled 1,3-butadiene with a range of simple dienophiles, allowing the endo:exo selectivities of these important reactions to be determined for the first time. The experimental data shed light on the origins of endo:exo selectivity in the Diels-Alder reaction and will serve as an important reference for future computational investigations in this area. The intramolecular Diels-Alder reaction shares many of the virtues of its intermolecular counterpart, however its use in chemical synthesis is limited because intramolecular Diels-Alder reactivity and stereoselectivity are often governed by subtle factors, and can be very difficult to predict. As part of a comprehensive experimental and computational collaboration, Chapter 3 describes an investigation of the heat and Lewis acid promoted intramolecular Diels-Alder reactions of some ether tethered 1,3,8-nonatrienes. Also presented are the results of a rate study and a kinetic isotope effect study involving the intramolecular Diels-Alder reactions of some 1,3,8-nonatrienes. The experimental data are analysed and compared with predicted stereoselectivities, activation barriers and kinetic isotope effects obtained from computational modelling. Increased efficiency in chemical synthesis conserves resources, reduces waste, and saves time and money. Domino reactions are particularly efficient processes, which can generate complex products from simple reactants. Chapter 4 describes an investigation of the domino Diels-Alder reactions of (1E,3E)-1,4-diiodo-1,3-butadiene with maleimide dienophiles, through which a family of bicyclo[2.2.2]oct-2-ene derivatives are produced in one high yielding and stereoselective synthetic step.

Organic semiconductors are a new key technology for large-area and flexible thin-film electronics. They are deposited as thin films (sub-nanometer to micrometer) on large-area substrates. The technologically most advanced applications are organic light emitting diodes (OLEDs) and organic photovoltaics (OPV). For the improvement of performance and efficiency, correct modeling of the electronic processes in the devices is essential. Reliable characterization and validation of the electronic properties of the materials is simultaneously required for the successful optimization of devices. Furthermore, understanding the relations between material structures and their key characteristics opens the path for innovative material and device design. In this thesis, two material characterization methods are developed, respectively refined and applied: a novel technique for measuring the charge carrier mobility μ and a way to determine the ionization energy IE or the electron affinity EA of an organic semiconductor. For the mobility measurements, a new evaluation approach for space-charge limited current (SCLC) measurements in single carrier devices is developed. It is based on a layer thickness variation of the material under investigation. In the \"potential mapping\" (POEM) approach, the voltage as a function of the device thickness V(d) at a given current density is shown to coincide with the spatial distribution of the electric potential V(x) in the thickest device. On this basis, the mobility is directly obtained as function of the electric field F and the charge carrier density n. The evaluation is model-free, i.e. a model for μ(F, n) to fit the measurement data is not required, and the measurement is independent of a possible injection barrier or potential drop at non-optimal contacts. The obtained μ(F, n) function describes the effective average mobility of free and trapped charge carriers. This approach realistically describes charge transport in energetically disordered materials, where a clear differentiation between trapped and free charges is impossible or arbitrary. The measurement of IE and EA is performed by characterizing solar cells at varying temperature T. In suitably designed devices based on a bulk heterojunction (BHJ), the open-circuit voltage Voc is a linear function of T with negative slope in the whole measured range down to 180K. The extrapolation to temperature zero V0 = Voc(T → 0K) is confirmed to equal the effective gap Egeff, i.e. the difference between the EA of the acceptor and the IE of the donor. The successive variation of different components of the devices and testing their influence on V0 verifies the relation V0 = Egeff. On this basis, the IE or EA of a material can be determined in a BHJ with a material where the complementary value is known. The measurement is applied to a number of material combinations, confirming, refining, and complementing previously reported values from ultraviolet photo electron spectroscopy (UPS) and inverse photo electron spectroscopy (IPES). These measurements are applied to small molecule organic semiconductors, including mixed layers. In blends of zinc-phthalocyanine (ZnPc) and C60, the hole mobility is found to be thermally and field activated, as well as increasing with charge density. Varying the mixing ratio, the hole mobility is found to increase with increasing ZnPc content, while the effective gap stays unchanged. A number of further materials and material blends are characterized with respect to hole and electron mobility and the effective gap, including highly diluted donor blends, which have been little investigated before. In all materials, a pronounced field activation of the mobility is observed. The results enable an improved detailed description of the working principle of organic solar cells and support the future design of highly efficient and optimized devices.:1. Introduction 2. Organic semiconductors and devices 2.1. Organic semiconductors 2.1.1. Conjugated π system 2.1.2. Small molecules and polymers 2.1.3. Disorder in amorphous materials 2.1.4. Polarons 2.1.5. Polaron hopping 2.1.6. Fermi-Dirac distribution and Fermi level 2.1.7. Quasi-Fermi levels 2.1.8. Trap states 2.1.9. Doping 2.1.10. Excitons 2.2. Interfaces and blend layers 2.2.1. Interface dipoles 2.2.2. Energy level bending 2.2.3. Injection from metal into semiconductor, and extraction 2.2.4. Excitons at interfaces 2.3. Charge transport and recombination in organic semiconductors 2.3.1. Drift transport 2.3.2. Charge carrier mobility 2.3.3. Thermally activated transport 2.3.4. Diffusion transport 2.3.5. Drift-diffusion transport 2.3.6. Space-charge limited current 2.3.7. Recombination 2.4. Mobility measurement 2.4.1. SCLC and TCLC 2.4.2. Time of flight 2.4.3. Organic field effect transistors 2.4.4. CELIV 2.5. Organic solar cells 2.5.1. Exciton diffusion towards the interface 2.5.2. Dissociation of CT states 2.5.3. CT recombination 2.5.4. Flat and bulk heterojunction 2.5.5. Transport layers 2.5.6. Thin film optics 2.5.7. Current-voltage characteristics and equivalent circuit 2.5.8. Solar cell efficiency 2.5.9. Limits of efficiency 2.5.10. Correct solar cell characterization 2.5.11. The \"O-Factor\" 3. Materials and experimental methods 3.1. Materials 3.2. Device fabrication and layout 3.2.1. Layer deposition 3.2.2. Encapsulation 3.2.3. Homogeneity of layer thickness on a wafer 3.2.4. Device layout 3.3. Characterization 3.3.1. Electrical characterization 3.3.2. Sample illumination 3.3.3. Temperature dependent characterization 3.3.4. UPS 4. Simulations 5.1. Design of single carrier devices 5.1.1. General design requirements 5.1.2. Single carrier devices for space-charge limited current 5.1.3. Ohmic regime 5.1.4. Design of injection and extraction layers 5.2. Advanced evaluation of SCLC – potential mapping 5.2.1. Potential mapping by thickness variation 5.2.2. Further evaluation of the transport profile 5.2.3. Injection into and extraction from single carrier devices 5.2.4. Majority carrier approximation 5.3. Proof of principle: POEM on simulated data 5.3.1. Constant mobility 5.3.2. Field dependent mobility 5.3.3. Field and charge density activated mobility 5.3.4. Conclusion 5.4. Application: Transport characterization in organic semiconductors 5.4.1. Hole transport in ZnPc:C60 5.4.2. Hole transport in ZnPc:C60 – temperature variation 5.4.3. Hole transport in ZnPc:C60 – blend ratio variation 5.4.4. Hole transport in ZnPc:C70 5.4.5. Hole transport in neat ZnPc 5.4.6. Hole transport in F4-ZnPc:C60 5.4.7. Hole transport in DCV-5T-Me33:C60 5.4.8. Electron transport in ZnPc:C60 5.4.9. Electron transport in neat Bis-HFl-NTCDI 5.5. Summary and discussion of the results 5.5.1. Phthalocyanine:C60 blends 5.5.2. DCV-5T-Me33:C60 5.5.3. Conclusion 6. Organic solar cell characteristics: the influence of temperature 6.1. ZnPc:C60 solar cells 6.1.1. Temperature variation 6.1.2. Illumination intensity variation 6.2. Voc in flat and bulk heterojunction organic solar cells 6.2.1. Qualitative difference in Voc(I, T) 6.2.2. Interpretation of Voc(I, T) 6.3. BHJ stoichiometry variation 6.3.1. Voc upon variation of stoichiometry and contact layer 6.3.2. V0 upon stoichiometry variation 6.3.3. Low donor content stoichiometry 6.3.4. Conclusion from stoichiometry variation 6.4. Transport material variation 6.4.1. HTM variation 6.4.2. ETM variation 6.5. Donor:acceptor material variation 6.5.1. Donor variation 6.5.2. Acceptor variation 6.6. Conclusion 7. Summary and outlook 7.1. Summary 7.2. Outlook A. Appendix A.1. Energy pay-back of this thesis A.2. Tables and registers
Organische Halbleiter sind eine neue Schlüsseltechnologie für großflächige und flexible Dünnschichtelektronik. Sie werden als dünne Materialschichten (Sub-Nanometer bis Mikrometer) auf großflächige Substrate aufgebracht. Die technologisch am weitesten fortgeschrittenen Anwendungen sind organische Leuchtdioden (OLEDs) und organische Photovoltaik (OPV). Zur weiteren Steigerung von Leistungsfähigkeit und Effizienz ist die genaue Modellierung elektronischer Prozesse in den Bauteilen von grundlegender Bedeutung. Für die erfolgreiche Optimierung von Bauteilen ist eine zuverlässige Charakterisierung und Validierung der elektronischen Materialeigenschaften gleichermaßen erforderlich. Außerdem eröffnet das Verständnis der Zusammenhänge zwischen Materialstruktur und -eigenschaften einen Weg für innovative Material- und Bauteilentwicklung. Im Rahmen dieser Dissertation werden zwei Methoden für die Materialcharakterisierung entwickelt, verfeinert und angewandt: eine neuartige Methode zur Messung der Ladungsträgerbeweglichkeit μ und eine Möglichkeit zur Bestimmung der Ionisierungsenergie IE oder der Elektronenaffinität EA eines organischen Halbleiters. Für die Beweglichkeitsmessungen wird eine neue Auswertungsmethode für raumladungsbegrenzte Ströme (SCLC) in unipolaren Bauteilen entwickelt. Sie basiert auf einer Schichtdickenvariation des zu charakterisierenden Materials. In einem Ansatz zur räumlichen Abbildung des elektrischen Potentials (\"potential mapping\", POEM) wird gezeigt, dass das elektrische Potential als Funktion der Schichtdicke V(d) bei einer gegebenen Stromdichte dem räumlichen Verlauf des elektrischen Potentials V(x) im dicksten Bauteil entspricht. Daraus kann die Beweglichkeit als Funktion des elektrischen Felds F und der Ladungsträgerdichte n berechnet werden. Die Auswertung ist modellfrei, d.h. ein Modell zum Angleichen der Messdaten ist für die Berechnung von μ(F, n) nicht erforderlich. Die Messung ist außerdem unabhängig von einer möglichen Injektionsbarriere oder einer Potentialstufe an nicht-idealen Kontakten. Die gemessene Funktion μ(F, n) beschreibt die effektive durchschnittliche Beweglichkeit aller freien und in Fallenzuständen gefangenen Ladungsträger. Dieser Zugang beschreibt den Ladungstransport in energetisch ungeordneten Materialien realistisch, wo eine klare Unterscheidung zwischen freien und Fallenzuständen nicht möglich oder willkürlich ist. Die Messung von IE und EA wird mithilfe temperaturabhängiger Messungen an Solarzellen durchgeführt. In geeigneten Bauteilen mit einem Mischschicht-Heteroübergang (\"bulk heterojunction\" BHJ) ist die Leerlaufspannung Voc im gesamten Messbereich oberhalb 180K eine linear fallende Funktion der Temperatur T. Es kann bestätigt werden, dass die Extrapolation zum Temperaturnullpunkt V0 = Voc(T → 0K) mit der effektiven Energielücke Egeff , d.h. der Differenz zwischen EA des Akzeptor-Materials und IE des Donator-Materials, übereinstimmt. Die systematische schrittweise Variation einzelner Bestandteile der Solarzellen und die Überprüfung des Einflusses auf V0 bestätigen die Beziehung V0 = Egeff. Damit kann die IE oder EA eines Materials bestimmt werden, indem man es in einem BHJ mit einem Material kombiniert, dessen komplementärer Wert bekannt ist. Messungen per Ultraviolett-Photoelektronenspektroskopie (UPS) und inverser Photoelektronenspektroskopie (IPES) werden damit bestätigt, präzisiert und ergänzt. Die beiden entwickelten Messmethoden werden auf organische Halbleiter aus kleinen Molekülen einschließlich Mischschichten angewandt. In Mischschichten aus Zink-Phthalocyanin (ZnPc) und C60 wird eine Löcherbeweglichkeit gemessen, die sowohl thermisch als auch feld- und ladungsträgerdichteaktiviert ist. Wenn das Mischverhältnis variiert wird, steigt die Löcherbeweglichkeit mit zunehmendem ZnPc-Anteil, während die effektive Energielücke unverändert bleibt. Verschiedene weitere Materialien und Materialmischungen werden hinsichtlich Löcher- und Elektronenbeweglichkeit sowie ihrer Energielücke charakterisiert, einschließlich bisher wenig untersuchter hochverdünnter Donator-Systeme. In allen Materialien wird eine deutliche Feldaktivierung der Beweglichkeit beobachtet. Die Ergebnisse ermöglichen eine verbesserte Beschreibung der detaillierten Funktionsweise organischer Solarzellen und unterstützen die künftige Entwicklung hocheffizienter und optimierter Bauteile.:1. Introduction 2. Organic semiconductors and devices 2.1. Organic semiconductors 2.1.1. Conjugated π system 2.1.2. Small molecules and polymers 2.1.3. Disorder in amorphous materials 2.1.4. Polarons 2.1.5. Polaron hopping 2.1.6. Fermi-Dirac distribution and Fermi level 2.1.7. Quasi-Fermi levels 2.1.8. Trap states 2.1.9. Doping 2.1.10. Excitons 2.2. Interfaces and blend layers 2.2.1. Interface dipoles 2.2.2. Energy level bending 2.2.3. Injection from metal into semiconductor, and extraction 2.2.4. Excitons at interfaces 2.3. Charge transport and recombination in organic semiconductors 2.3.1. Drift transport 2.3.2. Charge carrier mobility 2.3.3. Thermally activated transport 2.3.4. Diffusion transport 2.3.5. Drift-diffusion transport 2.3.6. Space-charge limited current 2.3.7. Recombination 2.4. Mobility measurement 2.4.1. SCLC and TCLC 2.4.2. Time of flight 2.4.3. Organic field effect transistors 2.4.4. CELIV 2.5. Organic solar cells 2.5.1. Exciton diffusion towards the interface 2.5.2. Dissociation of CT states 2.5.3. CT recombination 2.5.4. Flat and bulk heterojunction 2.5.5. Transport layers 2.5.6. Thin film optics 2.5.7. Current-voltage characteristics and equivalent circuit 2.5.8. Solar cell efficiency 2.5.9. Limits of efficiency 2.5.10. Correct solar cell characterization 2.5.11. The \"O-Factor\" 3. Materials and experimental methods 3.1. Materials 3.2. Device fabrication and layout 3.2.1. Layer deposition 3.2.2. Encapsulation 3.2.3. Homogeneity of layer thickness on a wafer 3.2.4. Device layout 3.3. Characterization 3.3.1. Electrical characterization 3.3.2. Sample illumination 3.3.3. Temperature dependent characterization 3.3.4. UPS 4. Simulations 5.1. Design of single carrier devices 5.1.1. General design requirements 5.1.2. Single carrier devices for space-charge limited current 5.1.3. Ohmic regime 5.1.4. Design of injection and extraction layers 5.2. Advanced evaluation of SCLC – potential mapping 5.2.1. Potential mapping by thickness variation 5.2.2. Further evaluation of the transport profile 5.2.3. Injection into and extraction from single carrier devices 5.2.4. Majority carrier approximation 5.3. Proof of principle: POEM on simulated data 5.3.1. Constant mobility 5.3.2. Field dependent mobility 5.3.3. Field and charge density activated mobility 5.3.4. Conclusion 5.4. Application: Transport characterization in organic semiconductors 5.4.1. Hole transport in ZnPc:C60 5.4.2. Hole transport in ZnPc:C60 – temperature variation 5.4.3. Hole transport in ZnPc:C60 – blend ratio variation 5.4.4. Hole transport in ZnPc:C70 5.4.5. Hole transport in neat ZnPc 5.4.6. Hole transport in F4-ZnPc:C60 5.4.7. Hole transport in DCV-5T-Me33:C60 5.4.8. Electron transport in ZnPc:C60 5.4.9. Electron transport in neat Bis-HFl-NTCDI 5.5. Summary and discussion of the results 5.5.1. Phthalocyanine:C60 blends 5.5.2. DCV-5T-Me33:C60 5.5.3. Conclusion 6. Organic solar cell characteristics: the influence of temperature 6.1. ZnPc:C60 solar cells 6.1.1. Temperature variation 6.1.2. Illumination intensity variation 6.2. Voc in flat and bulk heterojunction organic solar cells 6.2.1. Qualitative difference in Voc(I, T) 6.2.2. Interpretation of Voc(I, T) 6.3. BHJ stoichiometry variation 6.3.1. Voc upon variation of stoichiometry and contact layer 6.3.2. V0 upon stoichiometry variation 6.3.3. Low donor content stoichiometry 6.3.4. Conclusion from stoichiometry variation 6.4. Transport material variation 6.4.1. HTM variation 6.4.2. ETM variation 6.5. Donor:acceptor material variation 6.5.1. Donor variation 6.5.2. Acceptor variation 6.6. Conclusion 7. Summary and outlook 7.1. Summary 7.2. Outlook A. Appendix A.1. Energy pay-back of this thesis A.2. Tables and registers