Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.:1. Introduction............................................................................ 1 1.1. Anatomy and histology of the human urinary bladder..................... 1 1.1.1. Anatomy of the human urinary bladder..................................... 1 1.1.2. Structure of the human urinary bladder wall............................... 2 1.2. Normal bladder function and bladder dysfunction.......................... 3 1.2.1 Normal bladder function......................................................... 3 1.2.2 Sensory aspect.................................................................... 4 1.2.3 Overactivity or hypersensitivity of bladder.................................. 5 1.3 The role of functional cell types and interaction in urinary bladder... 6 1.3.1 The role of urothelium.......................................................... 7 1.3.2Theroleofsuburotheliamyofibroblast...................................... 7 1.3.3Theroleofdetrusorsmoothmusclecells.................................. 9 1.3.4 Possible interactions in urinary bladder cell types........................ 10 1.4 ATP function and Purinergic signalling in bladder........................... 11 1.5 Spontaneous activity of bladder................................................... 13 2. Objective.................................................................................. 15 3. Material and methods............................................................... 16 3.1. Ethics Statement........................................................................ 16 3.2. Cell preparation.......................................................................... 16 3.3. Solutions and chemicals............................................................. 19 3.4. Intracellular calcium measurements............................................. 20 2.4.1. Preparing cells for Calcium Imaging.......................................... 20 2.4.2. Preparing workspace of calcium imaging................................... 20 2.4.3. Calcium imaging recording...................................................... 22 3.5 Data analysis with automated Fluorescence analysis..................... 22 3.6 Confocal Immunofluorescence.................................................... 25 3.7 Statistics................................................................................. 26 4. Results.................................................................................. 27 4.1 Spontaneous calcium activity of sMF........................................... 27 4.2 ATP effects on calcium response in sMF...................................... 27 4.3 Analysis of purinergic receptors involved.................................... 30 3.3.1 Agonist stimulation.............................................................. 30 3.3.2 Signal inhibition by specific antagonists................................... 31 4.4 Confocal immunofluorescence of purinergic receptors.................. 32 5. Discussion............................................................................. 34 5.1 Myofibroblast identification....................................................... 34 5.2 Spontaneous activity in the bladder............................................ 36 5.3 ATP modulated calcium activity in sMF....................................... 37 5.4 purinergic signalling in sMF........................................................ 39 6. Summary................................................................................ 42 7. References.............................................................................. 45 Declaration............................................................................. 50 Acknowledgements................................................................. 51

Ce rapport présente des méthodes de prétraitement, d'analyse statistique et d'interprétation de chroniques hydrogéologiques de massifs peu perméables (argilites) dans le cadre d'études sur le stockage profond de déchets radioactifs. Les séries temporelles analysées sont la pression interstitielle et la pression atmosphérique, en relation avec différents phénomènes (marées terrestres, effet barométrique, évolution de l'excavation des galeries). Les pré-traitements permettent de reconstituer et homogénéiser les chroniques de données en présence de lacunes, aberrations, et pas de temps variables. Les signaux prétraités sont ensuite analysés en vue de caractériser les propriétés hydrauliques du massif peu perméable (emmagasinement spécifique ; porosité effective). Pour cela, on a développé et mis en oeuvre les méthodes d'analyses suivantes (implémentées en Matlab): analyses corrélatoires et spectrales (Fourier) ; analyses ondelettes multirésolution ; enveloppes de signaux aléatoires. Cette méthodologie est appliquée aux données acquises au Laboratoire Souterrain du Consortium International du Mont Terri (Jura Suisse), ainsi qu'à certaines données des Laboratoires Souterrains de Tournemire (Aveyron) et de Meuse / Haute-Marne (ANDRA) / This report presents a set of statistical methods for pre-processing and analyzing multivariate hydrogeologic time series, such as pore pressure and its relation to atmospheric pressure. The goal is to study the hydrogeologic characteristics of low permeability geologic formations (argilite) in the context of deep disposal of radioactive waste. The pressure time series are analyzed in relation with different phenomena, such as earth tides, barometric effects, and the evolution of excavated galleries. The pre-processing is necessary for reconstituting and homogenizing the time series in the presence of data gaps, outliers, and variable time steps. The preprocessed signals are then analyzed with a view to characterizing the hydraulic properties of this type of low permeability formation (specific storativity; effective porosity). For this sake, we have developed and used the following methods (implemented in Matlab): temporal correlation analyses; spectral/Fourier analyses; multiresolution wavelet analyses envelopes of random processes. This methodology is applied to data collected at the URL (Underground Research Laboratory) of the Mont Terri International Consortium (Swiss Jura), as well as some other data collected at the URL of IRSN at Tournemire (Aveyron) and at the URL of ANDRA (Meuse / Haute-Marne)