Systematic investigations through several experimental techniques have revealed that hippocampal pyramidal neurons express voltage gated ion channels (VGICs) with well-defined gradients along their dendritic arbor. These actively maintained gradients in various dendritic VGICs effectuate several stereotypic, topographically continuous functional gradients along the topograph of the dendritic arbor, and have been referred to as intraneuronal functional maps. The prime goal of my thesis was to understand the emergence and homeostasis of the several coexistent functional maps that express within hippocampal pyramidal neurons. In the first part of the thesis, we focus only on spatial interactions between ion channels and analyzed the role of such interactions in the emergence of functional maps. We developed a generalized quantitative framework, the influence field, to analyze the extent of influence of a spatially localized VGIC cluster. Employing this framework, we showed that a localized VGIC cluster could have spatially widespread influence, and was heavily reliant on the specific physiological property and background conductances. Using the influence field model, we reconstructed functional gradients from VGIC conductance gradients, and demonstrated that the cumulative contribution of VGIC conductances in adjacent compartments plays a critical role in determining physiological properties at a given location. These results suggested that spatial interactions among spatially segregated VGIC clusters are necessary for the emergence of the functional maps. In the second part of the thesis, we assessed the specific roles of only kinetic interactions between ion channels in determining physiological properties by employing a single-compartmental model. In doing this, we analyzed the roles of interactions among several VGICs in regulating intrinsic response dynamics. Using global sensitivity analysis, we showed that functionally similar models could be achieved even when underlying parameters displayed tremendous variability and exhibited weak pair-wise correlations. These results suggested that that response homeostasis could be achieved through several non-unique channel combinations, as an emergent consequence of kinetic interactions among these channel conductances. In the final part of the thesis, we analyzed the combined impact of both spatial and kinetic interactions among ion channel conductances on the emergence and homeostasis of functional maps in a neuronal model endowed with extensive dendritic arborization. To do this, we performed global sensitivity analysis on morphologically realistic conductance-based models of hippocampal pyramidal neurons that coexpressed six functional maps. We found topographically continuous functional maps to emerge from disparate model parameters with weak pair-wise correlations between parameters. These results implied that individual channel properties need not be set at constant values in achieving overall homeostasis of several coexistent functional maps. We suggest collective channelostasis, where several channels regulate their properties and expression profiles in an uncorrelated manner, as an alternative for accomplishing functional map homeostasis. Finally, we developed a methodology to assess the contribution of individual channel conductances to the various functional measurements employing virtual knockout simulations. We found that the deletion of individual channels resulted in variable, measurement-and location-specific impacts across the model population.

With an increased popularity for systems-based approaches in biology, a wide spectrum of techniques has been applied to the simulation and analysis of biochemical systems which involves uncertainty and stochasticity. It is particularly concerned with modelling and analysis of metabolic pathways, regulatory and signal transduction networks for understanding intra-cellular pathway behaviour. Typically, parameter estimation in ordinary differential equations(ODEs) models is used for this purpose when there is large number of molecules involved in the reaction system. However this approach is correct when the system is large enough to be deterministic in nature. But there are uncertainty involved in the system and the processes are stochastic in nature due to smaller population molecules participating in the pathway reactions. In this thesis the common theme is the study of uncertainties in the chemical kinetics of biochemical reaction systems associated with various intra-cellular pathways and channels. The study is at the mesoscale of the system, i.e., we study systems that do not have too few molecules disallowing any higher scale system level approximation nor too many where a non-stochastic (mesoscale) system approximation will be valid. In our first study we estimate the parameters in the mitogen activated protein kinase (MAPK) signal transduction pathway involved in the departure from the normal Epithelial Growth Factor(EGF) dose-dependency in prostate cancer cells. A model-based pathway analysis is performed. The pathway is mathematically modelled with 28 rate equations yielding those many ordinary differential equations(ODE) with kinetic rate constants that have been reported to take random values in the existing literature. This has led to us treating the ODE model of the pathways kinetics as a random differential equations(RDE) system in which the parameters are random variables. The most likely set of values of the kinetic rate constants obtained from fitting the RDE model into the experimental data is then used in a direct transcription based dynamic optimization method for computing the changes needed in these kinetic rate constant values for the restoration of the normal EGF dose response. It identifies the parameters, i.e., the kinetic rate constants in the RDE model, that are the most sensitive to the change in the EGF dose response behaviour in the PC3 prostate cancer cells. Biochemical pathways involving chemical kinetics equations in terms of low concen-trations of the model variables can be represented as chemical Langevin equations(CLE) as there is stochasticity involved in the processes. Most CLE systems come with the implicit constraint that the concentration state cannot be negative at any time over the sample path. Due to the inherent stiffness(especially in diffusion coefficient) of the CLE system, it has been difficult for numerical schemes to meet this positivity constraint during numerical simulations. Most available methods resort to heuristics by dropping selective noise terms from the original CLE inconsistent with the mesoscale physics involved in forming the CLE. Other methods take very small time steps thus making the simulation inefficient. In our second study we preserve positivity by using a physically consistent numerical scheme which is a modified form of fully stochastic α method for stiff stochastic differential equation. Ion channels are fundamental molecules in the nervous system that catalyse the flux of ions across the cell membrane. Single ion channel flux activity is comparable to the catalytic activity of single enzyme molecules. Saturating concentrations of substrate induce dynamic disorder in the kinetic rate processes of single enzyme molecules and consequently, develop correlative memory of the previous history of activities. Conversely, binding of substrate ion is known to alter the catalytic turnover of single ion channels. Here, we investigated the possible existence of dynamic disorder and molecular memory in single human TREK1 channel due to binding of substrate/agonist using the excised inside-out patch-clamp technique. Our results suggest that single hTREK1 channel behaves as a typical Michaelis-Menten enzyme molecule with a single high-affinity binding site for substrate K+ ion but with uncertainty in reaction rates.

This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel. In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.

La fibrillation atriale (FA) est l'arythmie cardiaque soutenue la plus fréquente chez les adultes. Elle est associée à une augmentation du risque d'accident vasculaire cérébral, d'insuffisance cardiaque et de la mortalité. Un mécanisme de torsades auriculaires a été décrit chez des patients atteints du syndrome du QT long congénital (SQTL). Malgré une littérature très riche sur le sujet, les mécanismes impliqués dans la genèse et le maintien de ces arythmies restent mal connus et constituent un obstacle dans le diagnostic et la prise en charge de ces maladies. Dans la première partie de ce travail, nous avons abordé l'histologie et la distribution des connexines (Cxs) chez deux groupes de patients avec et sans FA par une approche de microscopie confocale. Nous avons ainsi décrit d'une part un réseau entre fibroblastes et myocytes communiquant via les Cx 40 et 43 et d'autre part la présence de myofibroblastes, d'une forte fibrose et d'un remodelage des Cx 40 et 43 dans le tissu de patients FA. Par ailleurs, pour identifier de nouvelles mutations impliquées dans ces arythmies, nous avons étudié une cohorte de 60 patients atteints de FA. Les recherches génétiques et l'étude fonctionnelle nous ont permis d'associer 5 nouvelles mutations: P41S et M207V (PITX2), G277E (Cx 40) A253V (Cx 43) et P1034H (KCNH2) à la FA. Celles-ci semblent jouer un rôle clé dans la constitution du substrat arythmogène. Enfin, dans la dernière partie, nous avons exploré l'impact électrophysiologique d'un variant de KCNH2, R148W, trouvé tout d'abord chez un enfant décédé de mort subite pendant le sommeil, puis chez plusieurs membres de la famille, dont certains présentent un intervalle QT allongé. Ce variant, exprimé dans les ovocytes de Xénope et étudié en voltage-clamp, réduit le courant de 29% et pourrait alors prédisposer à la survenue de torsades de pointes et expliquer en partie l'allongement du QTc. Outre les nouveaux variants géniques découverts, ce travail est le premier à associer un gain de fonction du facteur de croissance PITX2c en relation avec la FA. Le remodelage histologique des Cx et les variants nucléotidiques touchant les gènes GJA1, GJA5, PITX2 et KCNH2 pourraient ainsi participer à l'étiologie de la FA et du QT long / Atrial fibrillation (AF) is the most common sustained arrhythmia in adults. It is associated with an increased risk of stroke, heart failure and mortality. A mechanism of atrial torsades has been described in patients with congenital long QT syndrome (LQTS). Despite the already existing body of literature, the mechanisms involved in the genesis and maintenance of these arrhythmias remain poorly understood and constitute an obstacle in diagnosis and management of these diseases. In the first part of this work, we discussed the histology of connexins (Cxs) and their distribution in two groups of patients (with and without FA), by confocal microscopy approach. We have described a network of fibroblasts and myocytes communicating across Cx 40 and 43 and the presence of myofibroblasts, of a strong fibrosis and of a remodeling of Cx 40 and 43 distribution in the tissue of AF patients. In addition, to identify new mutations involved in these arrhythmias, we studied a cohort of 60 patients with AF. Genetic investigations and functional study enabled us to associate five novel mutations with AF: M207V and P41S (PITX2), G277E (Cx 40) A253V (Cx 43) and P1034H (KCNH2). These mutations likely play a key role in the formation of the arrhythmogenic substrate. Finally, we explored the electrophysiological impact of a KCNH2 variant, R148W, initially found in a child who died suddenly during sleep and subsequently disclosed in several family members, some with a long QT interval. When expressed in Xenopus oocytes and studied in voltage-clamp, this variant reduces the current by 29%, which might predispose to torsades de pointes and partly explain the QTc prolongation. In addition to these newly discovered gene variants, this work is the first to report a gain-of-function mutation of the transcription factor PITX2c in AF. Histological remodeling of Cxs and the nucleotide variants affecting GJA1, GJA5, PITX2 and KCNH2 might thus participate in the etiology of AF and LQTS

No description available.

Inorganic ions are one of the most important group chemicals. Their importance is crucial for all living organisms on the planet and monitoring of their content is essential for this purpose in clinical or environmental analysis but also in the food industry, agriculture or many other industries. Many analytical methods for ion detection have been designed and used, depending on the area of their use. Recently, numerous methods have been adapted to areas where not primarily intended. One such methods may be, for example, laser ablation with inductively coupled plasma mass spectrometry, which has recently begun to affect the analysis of biological samples. As a complementary method, able to detect also non-metallic ions is capillary electrophoresis. In this master thesis, the possibility of applying capillary electrophoresis with indirect photometric detection for the separation of inorganic ions in cell lysates of HeLa cells by capillary electrophoresis was verified. Main attention was paid to chloride ions. The ability to separate inorganic compounds in cellular material using salicylic acid as an absorbing anion in the base electrolyte was tested.

Modification of Hodgkin and Huxley’s experimentally derived set of nonlinear differential equations was implemented to accurately simulate the action potential of the Hirudo Medicinalis’s Retzius cell in MATLAB under analogous conditions to those found in the Retzius cell environment. The voltage-gated sodium and potassium channel responses to changes in membrane potential, as experimentally determined by Hodgkin and Huxley, were manipulated to suit simulation parameters established by electrophysiological Retzius cell recordings. Application of this methodology permitted additional accurate simulation of the Hirudo Medicinalis’s P cell under analogous conditions to those found in the P cell environment. Further refinement of this technique should allow for the voltage-gated behavioral based simulation of action potential waveforms found in variety of neurons under simulation conditions analogous to the nerve cell environment.

The diploma thesis is focused on the analysis of the electrical properties of the heart cells membranes. The main goal of this work is to answer to what extent it is possible to infer from experimental data on the detailed mechanism of interaction of substances with channels. The work also presents a detailed description of methods of measurement of membrane voltage and current, which are used in the experimental measurements. There are also discussed the individual channels of the cell membrane and ion currents, which arise as a response to the rectangular pulse voltage. There is also described the effect of various substances on the properties of ion currents flowing through the cell membrane.

Les canaux ioniques ASIC (Acid-Sensing Ion Channel), largement exprimés dans le système nerveux central et périphérique, sont activés par une acidification extracellulaire. Ils sont impliqués dans un nombre croissant de fonctions physiologiques ou pathologiques telles que la transmission synaptique, la plasticité synaptique, l'apprentissage, la mémoire, la peur, la dépression, l’épilepsie et la neuro-dégénérescence, ainsi que la nociception et la mécanosensibilité. Ils sont des cibles thérapeutiques d’intérêt dans le traitement de la douleur ou de troubles neurologiques. Les toxines peuvent modifier le fonctionnement des canaux ioniques et sont par conséquent utiles pour l’étude de leurs structures et de leurs fonctions biologiques. Des médicaments élaborés à partir de toxines issues de venins ont également permis le traitement de nombreuses pathologies. Mon travail de thèse comporte deux axes. D’une part, la mise en place de nouvelles stratégies de collectes de venins avec la mise au point d’un nouvel extracteur électronique. Et d’autre part, l’identification du site de liaison de la mambalgine sur les canaux ASIC1a, la description de son mécanisme d’action ainsi que la caractérisation des résidus clefs de cette toxine. En se basant sur une nouvelle approche pharmacologique tirant partie des mécanismes d’action des toxines et des petites molécules nous avons pu établir un modèle plus global du mécanisme de régulation des ASIC par les protons. Ce travail ouvre des perspectives pour le développement de formes optimisées de la mambalgine et apporte de nouvelles informations sur les mécanismes d’activation et d’inactivation des canaux ASIC / The Acid-Sensing Ion Channels (ASIC) are widely expressed in the central and peripheral nervous system and are activated by extracellular acidification. They are involved in increasing number of physiological or pathological functions such as synaptic transmission, synaptic plasticity, learning, memory, fear, depression, epilepsy and neurodegeneration, as well as nociception and mechanical sensitivity. They represent interesting therapeutic targets for the treatment of pain and neurological disorders. Several toxins are able to modify the gating of ion channels and are useful for studying their structures and biological functions. Moreover, drugs developed from toxins venoms have allowed the discovery of new treatments for many pathologies. My thesis work has two axes. First, the development of a new electronic extractor has allowed the implementation of new strategies to collect venoms. On the other hand, we were able to identify the binding site of the mambalgine toxin on ASIC1a, to describe its mechanism of action and to characterize the key residues of this toxin. Based on a new pharmacological approach using action mechanisms of toxins and small molecules, we were able to establish a global model of the pH-dependent gating of ASIC channels. This work opens some new perspectives for the development of optimized forms of mambalgine and provides new information on the gating of ASIC

The stochastic behavior of voltage-gated ion channels causes fluctuations of conductances and voltages across neuronal membranes, contributing to the neuronal noise which is ubiquitous in the nervous system. While this phenomenon can be observed also on other parts of the neuron, here we concentrated on the axon and the way the channel noise influences axonal input-output characteristics. This was analysed by working with our newly created computational compartmental model, programmed in Matlab environment, built up using the Hodgkin-Huxley mathematical formalism and channel noise implemented via extended Markov Chain Monte Carlo method. The model was thoroughly verified to simulate plausibly a mammalian axon of CA3 neuron. Based on our simulations, we confirmed quantitatively the findings that the channel noise is the most prominent on membranes with smaller number of Na+ and K+ channels and that it majorly increases the variability of travel times of action potentials (APs) along axons, decreasing thereby the temporal precision of APs. The simulations analysing the effect of axonal demyelination and axonal diameter correlated well with other finding referred in Literature. We further focused on spike pattern and how is its propagation influenced by inter-spike intervals (ISI). We found, that APs fired...