Reg-1α est une protéine essentielle du système digestif impliquée dans des fonctions de prolifération, différenciation et régénération. Elle est exprimée et sécrétée par les neurones du système nerveux central où elle stimule la croissance neuritique et régule la différenciation et la migration des cellules précurseurs neurales via son récepteur EXTL3 et la voie GSK-3β. Par ailleurs, Reg-1α est surexprimée dans les cerveaux de patients Alzheimer et nos études préliminaires montrent qu’elle est associée à Tau hyperphosphorylée. Nous avons étudié dans ce contexte (1) le rôle de Reg-1α dans les processus neurodégénératifs liés à Tau et les voies de signalisation impliquées et (2) sa localisation dans le cerveau de souris transgéniques exprimant Tau mutée P301L/R406W. Nous avons montré que la surexpression de Reg-1α dans des neurones différenciés ne modifie pas significativement la voie Akt/GSK-3β/P-Tau mais induit la formation d’amas de Tau anormalement phosphorylée et une perturbation modérée du transport axonal. Par contre, sur des neurones surexprimant TauP301L, Reg-1α stimule la phosphorylation de Tau via Akt/GSK-3β entrainant la formation accentuée de renflements neuritiques et la perturbation sévère du transport axonal. In vivo, Reg-1α augmente avec l’âge dans le cerveau des souris contrôles et son expression est plus importante dès 5 mois chez les souris PLB2 Tau comparée aux souris sauvages. De plus, Reg-1α est associée à l’accumulation de Tau phosphorylée en S202 chez les souris transgéniques. Reg-1α est une protéine pouvant moduler l’hyperphosphorylation de Tau et le transport axonal et donc pourrait jouer un rôle dans le développement des tauopathies. / Reg-1α is an essential protein of the digestive system involved in proliferation, differentiation and regeneration functions. It is also expressed and secreted by neurons of the central nervous system where it stimulates neurite outgrowth and regulates differentiation and migration of neural precursor cells via its receptor EXTL3 and GSK-3β pathway. Moreover, Reg-1α is overexpressed in the brain of Alzheimer's patients and our preliminary studies show that it is associated with hyperphosphorylated Tau. We studied in this context (1) the role of Reg-1α in neurodegenerative processes associated with Tau and the involved signaling pathways and (2) its location in the brain of transgenic mice expressing mutated Tau P301L/R406W (PLB2 mice). We showed that overexpression of Reg-1α in differentiated neurons does not significantly modify the Akt/GSK-3β/P-tau pathway. However it induces the formation of neuritic swellings associated with abnormal phosphorylated Tau and leads to the disruption of axonal transport. Furthermore, in neurons overexpressing TauP301L, Reg-1α overexpression stimulates Tau phosphorylation via Akt/GSK-3β regulation and results in the increase of neuritic bulges and severe disruption of axonal transport. In vivo, Reg-1α expression increases with age in brains of control mice and is already higher in 5 month-old PLB2 Tau mice compared with age-matched controls. Cellular localization showed that Reg-1α is associated with the accumulation of phosphorylated Tau S202 in PLB2 mice. Finally, Reg-1α can regulate Tau hyperphosphorylation and axonal transport and consequentely could be involved in Tauopathy development.

There have been a number of computational modelling studies that aim to replicate the cerebellar Purkinje cell, though these typically use the morphology of rodent cells. While many species, including rodents, display intricate dendritic branching, it is not a universal feature among Purkinje cells. This study uses morphological reconstructions of 24 Purkinje cells from seven species to explore the changes that occur to the cell through evolution and examine whether this has an effect on the processing capacity of the cell. This is achieved by combining several modes of study in order to gain a comprehensive overview of the variations between the cells in both morphology and behaviour. Passive and active computational models of the cells were created, using the same electrophysiological parameters and ion channels for all models, to characterise the voltage attenuation and electrophysiological behaviour of the cells. These results and several measures of branching and size were then used to look for clusters in the data set using machine learning techniques. They were also used to visualise the differences within each species group. Information theory methods were also employed to compare the estimated information transfer from input to output across each cell. Along with a literature review into what is known about Purkinje cells and the cerebellum across the phylogenetic tree, these results show that while there are some obvious differences in morphology, the variation within species groups in electrophysiological behaviour is often as high as between them. This suggests that morphological changes may occur in order to conserve behaviour in the face of other changes to the cerebellum.

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Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kryštof Dobečka Supervisor: PharmDr. Tomáš Smutný, Ph.D. Advisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Experimental approaches for studying hepatic enzyme induction mediated by pregnane X receptor The thesis focuses on hepatic pregnane X receptor (PXR)-mediated induction of biotransformation enzymes. Emphasis is placed on experimental models and methods which are used for the assessment of enzyme induction. In addition to summarizing its well- established role as a xenobiotic-sensing receptor, PXR is also presented as a transcription factor with an important role in endogenous pathways. Furthermore, cell and animal models are evaluated in terms of expression and function of PXR and its target xenobiotic-metabolising enzymes. Primary human hepatocytes in 2D cultures are considered to be the gold standard of in vitro hepatic models. However, 3D technologies are expected to be increasingly used in the future. The use of animal models is limited due to pronounced interspecies differences in PXR activation. Thus, humanized models have been established to overcome these limitations. Next, this thesis comments screening methods for an assessment of interaction between PXR and...

Aujourd'hui, un grand nombre de nouvelles molécules peuvent être synthétisées à partir de la biomasse. Les tensioactifs dérivés de sucre sont notamment considérés comme une alternative aux tensioactifs fossiles en raison de leur biodégradabilité et de leur biocompatibilité. Cependant, les études associant la caractérisation physico-chimique et les propriétés biologiques de ce type de tensio-actifs sont limitées. Il est ainsi difficile de prédire les propriétés d'un tensioactif à partir de sa structure chimique. L'établissement d'une méthodologie permettant de relier la structure des surfactants à leurs propriétés apparait pertinent. Dans ce travail, quatre surfactants dérivés de sucre ayant chacun une chaîne C8 liée à une tête glucose ou maltose par un groupe amide ont été caractérisés par leurs propriétés tensio-actives dans différentes solutions (eau et milieu biologique). Leurs interactions avec des protéines ont également été analysées. Concernant l'évaluation des propriétés biologiques, des tests de cytotoxicité/irritation ont été effectués sur trois modèles in-vitro : 1) modèle cellulaire 20 (cellules L929 cultivées en monocouche), Il) modèle cellulaire 30 (cellules L929 cultivées dans un gel de collagène), Ill) épiderme humain reconstitué. Les résultats indiquent que les quatre surfactants synthétisés présentent de bonnes propriétés tensio-actives et trois d'entre eux sont moins cytotoxiques que des tensioactifs de référence. Plusieurs hypothèses permettant de relier la structure chimique des molécules à leurs propriétés physico-chimiques et biologiques ont été proposées. Des travaux futurs permettront d'enrichir la base de données sur les relations structure-propriétés des tensioactifs issus de la biomasse, et de l'utiliser pour synthétiser des surfactants présentant des propriétés adaptées aux applications envisagées. / Nowadays, a wide variety of new molecules can derive from biomass. Among them, the family of sugar-based surfactants, which are considered as alternatives to fossil-based surfactants, due to their relatively high biodegradability and biocompatibility, exhibit interesting properties both in terms of their self-assembly and their ability to induce biological responses. In the study, for the purpose to analyse these properties, different methodologies have been established. In this work, physico-chemistry and cellular biology methodologies are associated to analyse the properties of pre-selected molecules characterized by gradua) structure modifications. Firstly, we have screened synthesized sugar-based surfactants according to their solubility and their ability to reduce surface tension of water. Four pre-selected molecules, with a C8 chain linked to a glucose or maltose head through an amide functional group, either under the form of carbamoyl (carbohydrate scaffold bearing the carbonyl) or alkylcarboxamide (the alkyl chain bearing the carbonyl), were then dissolved in water/ cell culture media for surface tension measurements. Their behaviors in solutions were characterized by Krafft points, Critical Micellar Concentrations or self-assembling properties through different methods. To evaluate the cytotoxic/ irritant effects of these molecules on cells and tissues, 3 in-vitro models were established: I) 2D cell culture mode! (L929 cell monolayer) II) 3D ce!! culture mode! (L929 cells embedded in collagen gel) and III) Reconstituted human epidermis (differentiated human keratinocytes). Corresponding experiments were carried out on these models with increasing complexity. Results show that the synthesized sugar-based surfactants, GlulamideC8, Glu6amideC8, Glu6amideC8' and MallamideC8 can reduce the surface tension of water solution to the came level as standard surfactants (Tween 20 and Hecameg). In the meantime, GlulamideC8, Glu6amideC8' and MallamideC8 present Iess cytotoxicity effects on L929 cells both in the monolayer model and the 3D mode! than Tween 20 and Hecameg. All synthesized and standard surfactants (GlulamideC8, Glu6amideC8, Gu6amideC8', MallamideC8, Tween 20 and Hecameg) have no significant cytotoxic/ irritant effects on reconstituted human epidermis at 1000 ig/mL after 48 h of topical application. Discussions have been made according to the results of experiments to establish possible structures/ physico-chemical properties - cytotoxicity relationships of these surfactants.

The theoretical part of the thesis deals with the explanation of the actions that occur during the stimulation of tissues with the electric current. A significant analogy with electrical circuits is used to describe the phenomena at the molecular and cellular level. The models of membrane and cell are necessary for understanding the behaviour of more complex structures, such as tissues and organs. A considerable attention is paid to the conditions of electrical stimulation which bring about response in the stimulated area. Next, the cumulative effect of the subthreshold stimulation is analysed. The mechanisms of common treatment effects of the electrotherapeutic methods are outlined. The research results in the practical part of the thesis – the design for a microstimulator. Properties of the microstimulator and compliance with standard requirements are verified by testing the electromagnetic compatibility and electrical safety, conducted by the Institute for testing and certification, JSC. The effects of microstimulation on living organisms are experimentally investigated on horses, in collaboration with the Veterinary and Pharmaceutical University. For the first time, thermodynamic sensors are used for the objective assessment of the microstimulation therapeutic effect. These miniature sensors are placed on the horse´s front legs and monitor the changes in thermal activity while only one limb is really stimulated and the other is just considered as a reference. Comparison and statistical evaluation of the measured signals could provide a more detailed view of the thermal changes within the stimulated area, which is significantly related to blood circulation in limbs, and with the support of the reduction of edema. The course of the experiment which deals with the effect of microstimulation on edema of the horse´s legs caused by minor injuries (tendinitis, sprains, etc.), is documented in photographs or videos that are significant for possible evaluation of the effectiveness of the stimulation in this application.