Les vésicules sont des structures auto-assemblées formant des compartiments clos à l’échelle nanométrique ou micrométrique. Les différentes applications envisagées pour ces objets vont de la vectorisation de principes actifs à leur utilisation en tant que nano/micro-réacteur. Elles nécessitent donc un contrôle fin des propriétés de perméabilité des membranes vésiculaires. Au cours de cette thèse, des vésicules dont les membranes sont formées par des copolymères à blocs amphiphiles, vésicules appelées polymersomes, ont été formulées et étudiées. Pour répondre au mieux aux applications mentionnées, nous avons synthétisé de nouveaux copolymères amphiphiles capables de s’auto-assembler sous forme de vésicules pour étudier l’influence de l’architecture moléculaire sur les propriétés de membrane. Différentes stratégies pouvant permettre de moduler la perméabilité de ces assemblages ont ainsi été mises au point et étudiées : la formation de membranes hybrides polymère / lipide pour l’eau et l’utilisation d’un transporteur ionique pour les ions calcium. Finalement, l’influence de l’architecture macromoléculaire du copolymère sur l’évolution de forme des vésicules sous l’action d’un stress osmotique a été déterminée. Cette étude, alliant des analyses par diffusion de lumière et de neutrons ainsi que des études en microscopie électronique, a permis d’établir une modélisation complète. / Vesicles are self-assembled structures which form closed compartments at the nanometric or micrometric scale. These synthetic objects can be used for several kinds of applications, ranging from drug delivery to nano / microreactor for confined chemical reactions. The need to precisely control the vesicle membrane permeability is common to all these applications. In this thesis, vesicles resulting from the self-assembly of amphiphilic block copolymers, namely polymersomes, were prepared and studied. For this purpose, we synthesized new amphiphilic copolymers that can self-assemble into vesicles to study the influence of macromolecular parameters on the membrane properties. The formation of polymer / lipid hybrid vesicles for the water permeability and the use of an ion carrier selective for divalent cations have been studied as different methods which should allow the tuning of the membrane permeability. Finally, the influence of the copolymer architecture, molecular weight and hydrophobic chemical nature on the shape evolution of vesicles submitted to an osmotic stress has been studied. This study, combining light and neutron scattering with electron microscopy, allowed us to establish a complete modelisation of the shape evolution trajectory.

Neurons in the Locus coeruleus (LC) play an important role in the central CO2 chemosensitivity. However, the molecular mechanisms for neuronal CO2 chemosensitivity remain unclear. To demonstrate the expression of pH/CO2 sensitive ion channels, we screened the inward rectifier K+ channels (Kir) and transient receptor protein (TRP) channels, as parallel studies in this lab suggested that certain Kir and TRP channels are involved in neuronal responses to high levels of CO2. Our results showed that several members of the Kir and TRP channel families were robustly expressed in the LC neurons at the mRNA level. Of particular interest are TRPC5, Kir4.1 and Kir5.1 channels that are all pH-sensitive. The rich expression of various pH-sensitive Kir and TRP channels suggests that these ion channels are likely to play a role in the chemosensitivity of LC neurons.

Patients with Rett Syndrome (RTT) show severe breathing disorders in addition to other neuropathological features, contributing to the high incidence of sudden unexplained death and abnormal brain development. However, the molecular and cellular mechanisms underlying the breathing disorders are still unknown. Recent studies indicate that the dysfunction of brainstem norepinephrine (NE) systems are closely associated with breathing disorders in RTT patients as well as its mice model, the Mecp2-null (Mecp2─/Y) mice. This as well as the fact the major group of NE-ergic neurons in the locus coeruleus (LC) is CO2 chemosensitive suggests that the breathing disorders in RTT may be related these LC neurons. To test this hypothesis, we took a multidisciplinary approach and systematically studied these neurons using molecular biology, in-vitro brain slices, acutely dissociated neurons, immunocytochemistry, and whole-body plethysmograph. To facilitate the electrophysiological studies, we developed a new strain of transgenic mice with GFP expression selectively in the LC neurons of both WT and Mecp2─/Y mice. Breathing activity of the Mecp2─/Y mice showed selective disruptions in responses to mild hypercapnia. The defect was alleviated with the NE uptake blocker desipramine, suggesting the involvement of NE in central CO2 chemosensitivity. In the LC region, the expressions of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) at both protein and mRNA levels reduced by ~50% in Mecp2─/Y mice. No evidence was found for selective deficiency in TH- or DBH-containing neurons in Mecp2─/Y mice, and no major loss of NE-ergic LC cells were found, indicating that the NE defect is likely to result from deficient expression of biosynthetic enzymes rather than a loss of neurons in the LC. Several intrinsic membrane properties were abnormal in Mecp2─/Y LC neurons in comparison to wild type cells, including stronger inward rectification, shorter time constant, extended action potential duration, smaller amplitude of medium afterhyperpolarization (AHP) and over-expression of fast AHP. These abnormalities seem to be associated with the altered K+ and Na+ currents. Most importantly, Mecp2─/Y LC neurons displayed defective CO2 chemosensitivity in agreement of in vivo CO2 response, likely due to excessive expression of the homomeric Kir4.1 channel. Thus, it seems that the global effect of MeCP2 on the A6 NE system contributes to the impaired systemic CO2 response as well as the breathing irregularities in Mecp2─/Y mice. Such an alteration allowed CO2 to be detected only when hypercapnia became severe, leading to periodical hyper- and hypoventilation. These findings not only provide a novel etiology for the breathing disturbances of Mecp2─/Y mice but also show direct evidence for the first time on a molecular mechanism for the central CO2 chemosensitivity.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder of the central nervous system characterized by loss of motor neurons and voluntary muscle degeneration. Astrocytes play a major role in regulation of the disease onset and progression due to their intimate association with neurons. Regulation of ionic homeostasis is one of their key functions and its failure has been linked to several neurological diseases. The aim of this thesis was to explore differences in membrane properties of astrocytes in ALS. To fulfill this aim, a double transgenic mouse strain with ALS-like phenotype and a specific expression of enhanced green fluorescent protein in astrocytes was generated. To phenotype this strain, two sensorimotor tests, wire grid hang test and rotarod test, were conducted. Immunohistochemistry was used to characterize the strain on a cellular level and to explore changes of specific ion channels. Functional properties of astrocytes were explored using the patch clamp technique. The double transgenic strain has the characteristic ALS-like phenotype and is comparable to the original strain with differences in symptom onset and progression between models and sexes. On the cellular level, there are characteristic ALS features, specifically loss of motor neurons and astrogliosis....

Diese Arbeit beschäftigt sich mit zwei verschiedenen Modellsystemen, die Aufschluss über die Mechanik und die Dynamik von zellulären Systemen geben sollen. Zum Einsatz kommt zum einen der Modellorganismus Dictyostelium discoideum, dessen kollektives Migrationsverhalten analysiert wird und zum anderen wird die Mechanik von aktingefüllten Riesenvesikeln als artifizielles Modellsystem etabliert.