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Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Klára Dohnalová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Inhibitory effect of SPA70 on hPXR activation This work focuses on pregnane X receptor (PXR) and its antagonists. PXR is a ligand-activated nuclear receptor that plays a major role in detoxification of xenobiotics and protecting the organism from their toxic effects. Recent evidence also shows endogenous action of PXR in the metabolism of lipids, glucose and bile acids. However, PXR activation could be harmful, since induction of biotransformation enzymes by PXR agonists may result in reduced treatment efficacy, increased toxicity of drug metabolites and resistance to chemotherapeutic agents. Recent research has been intensively focused on PXR antagonists capable of abolishing these unfavourable effects. Recently discovered human PXR antagonist SPA70 has a promising potential for future usage. In this study, we investigated the inhibitory effect of SPA70 on activated PXR. To activate PXR we used agonists binding directly to PXR (rifampicin, hyperforin, SR12813) and also agonists activating PXR indirectly via cell signalling pathways (U0126, PD184352, PD0325901). Experiments were performed using luciferase...

Cellulose is a biopolymer of great relevance in the plant cell walls, where it constitutes the most important skeletal component. Cellulose is also an important raw material in the pulp- and paper, forest, and textile industries, among others. Cellulose biosynthesis in particular, and xylogenesis in general are processes which are currently poorly understood. Yet, research in cellulose synthesis is progressing and different applications of cellulose, mainly cellulose derivatives for e.g. pharmaceuticals and coatings, are constantly emerging. This thesis depicts how cellulose synthase (CesA) genes in Populus were identified and characterized by gene expression- and bioinformatics analyses. Within an EST database of more than 100,000 clones from wood forming tissues of three different Populus taxa, ten CesA genes were identified in Populus tremula x tremuloides. Subsequent gene expression analyses by using microarrays and real-time PCR experiments in woody tissues, revealed distinct regulation patterns among the genes of interest. This enabled proper classification and characterization of the secondary cell wall related CesA genes, in particular. Bioinformatic analyses of the genome sequence of Populus trichocarpa further provided a complete picture of the number of putative CesA genes retained after several duplication events during tree evolution. In contrast to the previously reported set of ten 'true' CesA genes in many other plant species, the genome of P. trichocarpa encodes 18 putative proteins, which could be assembled into nine groups according to their sequence similarities. Interestingly, studies in the EST database suggested that paralogs within at least two groups have corresponding orthologs in P. tremula x tremuloides, which are furthermore transcribed. This implies that at least some of the duplicated genes have remained functional, or may have acquired a modified function. By focusing on the CesA genes associated with secondary cell wall formation, cellulose synthesis was also studied in poplar cell suspension cultures. Selection of CesA enriched material was performed by determining expression intensities of the CesA genes using RT-PCR, whereupon membrane protein extraction was initiated. CesA proteins are part of large cellulose synthesizing complexes in the plasma membrane. Subsequent proteomic approaches comprised partial purification of these cellulose synthesizing complexes from protein enriched culture material and in vitro cellulose synthesis experiments. De novo synthesized material was successfully characterized and the acquired yields were as high as 50% cellulose (compared to previously reported yields of 30% in other plant systems) of the total in vitro synthesized product. Elevated CesA gene expression levels can thus be correlated to increased protein activity in poplar cell suspension cultures. In addition, antibodies raised against CesA antigens were used in Western blot analyses comprising samples along the protein extraction- and purification procedure. Proteins with corresponding molecular weight to the theoretical 120kDa of CesA proteins were recognized by a range of different specific antibodies. The study demonstrates that poplar cell suspension cultures can provide a valuable model system for studies of cellulose synthesis and different aspects of xylogenesis. / QC 20101005

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La Leucémie Lymphoïde Chronique (LLC) est caractérisée par l’accumulation dans le sang de lymphocytes B/CD5+ qui sont déficients pour leur programme de mort apoptotique. Malgré les progrès thérapeutiques récents, la LLC est une maladie toujours incurable. La stratégie de rétablissement des processus apoptotiques semble pertinente pour améliorer les traitements des patients, et cette stratégie nécessite l’identification de cibles spécifiques. Dans ce but, nous avons développé une recherche du mécanisme d’action de l’hyperforine, un phloroglucinol purifié à partir de la plante Millepertuis, qui est un inducteur d’apoptose des cellules de LLC ex vivo par la voie mitochondriale dépendante des caspases. Les résultats montrent que le traitement des cellules primaires de LLC régule positivement Noxa, une protéine de la famille Bcl-2 du type « BH3-only » qui est un ligand antagoniste spécifique de Mcl-1, la protéine de survie majeure pour le défaut d’apoptose dans les LLC. Nous montrons que Noxa interagit effectivement avec Mcl-1, ce qui entraine la dissociation du complexe que Mcl-1 forme avec Bak, et l’activation de cette protéine responsable de la libération par la mitochondrie des facteurs d’activation des caspases. La régulation positive de Noxa ne résulte apparemment pas d’une activation transcriptionnelle mais plutôt d’une régulation au niveau protéique comme le suggère la capacité de l’hyperforine à inhiber l’activité protéolytique du protéasome dans les cellules de LLC. Par ailleurs, l’apoptose induite par l’hyperforine est réduite en partie par l’extinction de Noxa avec des ARN interférents. Nos résultats indiquent donc que la régulation positive de Noxa est un des mécanismes qui permet à l’hyperforine de déclencher l’apoptose des cellules de LLC. Ce mécanisme semble être commun à d’autres composés d’origine végétale, comme nous l’avons observé avec l’extrait M2Yn dans la seconde partie de notre travail qui a permis de mettre en évidence que cet extrait de plantes d’Asie centrale est capable lui aussi d’induire l’apoptose des cellules de LLC par la voie mitochondriale dépendante des caspases. Enfin, nous avons contribué à la mise en évidence des propriétés pro-apoptotiques de l’hyperforine dans un autre modèle de leucémie, la leucémie myéloïde aiguë, grâce en partie à la régulation positive de Noxa ainsi qu’à l’inhibition de l’activité kinasique de Akt-1.En conclusion, nos travaux de thèse décrivent des nouvelles étapes dans le mécanisme d’action de l’hyperforine et permettent d’établir que la protéine « BH3-only » Noxa est une cible majeure pour la stratégie thérapeutique du rétablissement d’apoptose dans les LLC, ce qui devrait susciter le développement de nouveaux agents capables d’imiter spécifiquement l’action de Noxa. / Chronic lymphocytic leukemia (CLL) is characterized by blood accumulation of CD5+/B lymphocytes which are deficient in their apoptotic program. Despite recent therapeutic advances, CLL remains an incurable disease. Treatment strategy based on the re-establishment of apoptotic processes has retained much attention, and such a strategy requires the identification of specific targets. To this aim, we have investigated the mechanism of action of hyperforin, a phloroglucinol purified from St John’s wort, capable of inducing apoptosis of CLL cells ex vivo via the caspase-dependent mitochondrial pathway. Our results show that treatment of CLL patients’ cells upregulates the BH3-only protein Noxa that is a specific antagonist ligand of Mcl-1, the crucial prosurvival protein for apoptosis deficiency in CLL. We find that hyperforin provokes both the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/Bak complex, leading to Bak activation responsible for the release of apoptogenic factors from mitochondria. Noxa upregulation does not seem to result from transcription activation but rather from a regulation at the protein level, as suggested by the ability of hyperforin to inhibit proteasome activity in CLL cells. Using siRNA, Noxa silencing partially reduces hyperforin-induced apoptosis. Our data indicate that Noxa upregulation is one of the mechanisms through which hyperforin triggers CLL cell apoptosis. This mechanism appears to be shared with other plant-derived compounds, as observed with M2Yn, an extract from central Asia plants, that we show here to be an inducer of apoptosis in CLL cells by activating the caspase-dependent mitochondrial pathway. Finally, we have participated in highlighting the proapoptotic properties of hyperforin in another leukemia model, acute myeloid leukemia, via in part Noxa upregulation and Akt1 kinase activity inhibition.In conclusion, our data describe new steps in the mechanism of action of hyperforin and favor that the BH3-only protein Noxa is a major target for the therapeutic strategy based on apoptosis induction in CLL. Thus, this thesis should prompt the development of new BH3 mimetics specific for Noxa.

Due to various shortcomings of the visual system, some visual stimuli can only be identified with 100% accuracy if they are shown for a certain amount of time. This time can be measured using the Inspection Time (IT) paradigm. In an IT task, a “pi” figure with differing leg lengths is typically presented briefly (e.g., 20-200 ms) and is then immediately masked to prevent retinal afterimages. Participants are subsequently required to choose which of the two legs was longer. The objective of this task is to determine the shortest amount of time the pi figure needs to be shown for it to be perceived with 80% accuracy. Given that visual processing has been shown to be altered during and /or prior to a movement, the present experiment sought to test how the requirement to perform a motor task affected IT. Twenty-eight participants took part in the experiment, which was comprised of three conditions: no-movement (NM), peak velocity (PV), and foreperiod (FP). In the NM condition, participants grasped a manipulandum and engaged in the IT paradigm. At the end of every trial, participants verbally stated which leg they believed was longest. In the PV condition participants made a rapid movement to a target, and the IT stimulus was presented when their limb reached peak velocity. Finally in the FP condition the IT stimulus was presented during foreperiod (FP). In all three conditions the IT stimulus was randomly presented from between 15-105 ms (in 15 ms increments) and masked for 400 ms. Results showed no significant differences on the IT task between the NM and FP conditions, suggesting no visual upregulation during foreperiod. However, IT performance was significantly poorer in the PV condition in comparison to both the NM and FP condition, suggesting a visual downregulation at that particular movement kinematic.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lenka Linhartová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Molecular mechanisms of intractions - interactions of constitutive androstane receptor with selected stilbene compounds Constitutive androstane receptor (CAR), member of nuclear receptors family, is a major regulator of gene expression of phase I and II enzymes metabolizing endobiotics and xenobiotics. Changes in its activity can lead to pharmacokinetic drug interactions, ineffective treatment or higher toxicity of drugs simultaneously administered with CAR ligands. Recently another effects of this receptor, especially in homeostasis of bile acids, lipids and glucose have been discovered and CAR is now considered as a potential drug target for the treatment of metabolic diseases. Stilbenes represent a small group of plant polyphenols with typical 1,2-diphenylethylene nucleus. The most famous member is resveratrol, which has attracted great attention thanks to its antioxidant, anti-inflammatory, antiproliferative and cardioprotective effects. Others stilbene compounds such as pterostilben, piceatannol or pinosylvin have shown similar health beneficial effects as well. The aim of this diploma thesis was...

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