Le système nerveux est un réseau complexe qui détecte et analyse les informations. Ces informations sont transmises entre cellules grâce à des connections synaptiques et des jonctions communicantes. Ce réseau n’est pas statique et évolue au cours du développement, de l’apprentissage mais aussi durant le processus de vieillissement – naturels ou pathologiques. Comprendre le système nerveux et son fonctionnement requiert une analyse des connections synaptiques in vivo chez un animal model tel que Caenorhabditis elegans. Cependant les techniques actuellement disponibles pour C. elegans sont laborieuses, ne dépendent pas forcément d’une interaction trans-synaptique ou fixent la synapse. Par conséquent, ces approches ne permettent pas de réaliser des études de populations et dynamiques des modifications synaptiques. Dans ce manuscrit, je décris tout d’abord une nouvelle technique pour visualiser les synapses in vivo chez le vers C. elegans. Cette technique appelé iBLINC (in vivo Biotin Labeling of INtercellular Contacts) qui consiste en la biotinylation d’un peptide par une ligase d’Escherichia coli, BirA. Ces deux molécules sont fusionnées à des protéines trans-membranaires qui forment un complexe à la synapse. La biotinylation est détectée grâce à une streptavidin monomérique taguée avec un fluorophore qui est secrétée dans l’espace extracellulaire. J’ai démontré que cette technique est directionnelle et dynamique. En utilisant iBLINC pour visualiser des synapses faisant partie du circuit sensoriel de C. elegans, une évolution du nombre et de la taille des synapses a pu être observée avec l’âge. Il semblerait que ce changement soit dépendant du segment de la zone synaptique observée. Ces résultats ont été corroborés par l’observation d’une diminution du nombre de vésicules pendant le vieillissement grâce à un marqueur pré-synaptique des synapses GABAergique de la jonction neuromusculaire. Pour conclure, ce manuscrit décrit une nouvelle technique permettant d’observer les synapses chez le vers vivant et démontre une évolution naturelle du nombre de synapses et du nombre de vésicules pré-synaptiques avec l’âge. / The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates as it is involved in behavior responses. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections. Importantly, the network is not static and is believed to change during development and learning, as well as during pathological or normal age-related decline. Studying the nervous system in vivo requires the use of animal models such as Caenorhabditis elegans. Understanding of behavior and development requires visualization and analysis of synaptic connectivity. However, existing methods are laborious and may not depend on trans-synaptic interactions, or otherwise ‘trap’ the synapses by fixing the connections, thus precluding dynamic studies. In order to study synaptic modifications, we developed a new transgenic approach for in vivo labeling of specific connections in C. elegans, called iBLINC (in vivo Biotin Labeling of INtercellular Contacts). iBLINC involves the biotinylation of an acceptor peptide (AP) by the Escherichia coli biotin ligase BirA. Both are fused to two interacting post- and pre-synaptic proteins, respectively. The biotinylated acceptor peptide fusion is detected by a monomer streptavidin fused to a fluorescent protein that is transgenically expressed and secreted into the extracellular space. The method is directional, bright and dynamic. Moreover it correlates well with electron micrograph reconstruction. Using this new technique to observe synapses, which are part of the thermosensory circuitry of C. elegans, during aging, we could conclude that the connection pattern varies with age and within a population. Changes of the number and size of the synapses were observed during aging. Some segments of the synaptic region seem to be more affected than others by the aging process. Those results were corroborated by using a GABAergic pre-synaptic marker which allowed us to visualize a decline of the vesicle number with aging. In summary, in this thesis I explained a new in vivo trans-synaptic method to visualize synapses in C. elegans. Then I demonstrated that a natural decline in the number of synapses as well as the number of vesicles occurs during aging.

Le stress oxydant, les dysfonctions mitochondriaux et les altérations du métabolisme lipidique sont un dénominateur commun des maladies neurodégénératives (MN), comme la sclérose en plaques (SEP). Dans les conditions du stress oxydant, l’excès du cholestérol est éliminé par oxydation, produisant des oxystérols. Chez l'homme, le taux du 7β-hydroxycholestérol (7β-OHC) est souvent trouvé à des taux élevés dans le liquide céphalo-rachidien (LCR) et/ ou le plasma de patients atteints de MN, notamment la SEP.Dans ce contexte, une étude clinique préliminaire sur le LCR et le plasma de patients atteints de SEP RR a été réalisée dans le but de rechercher des biomarqueurs du métabolisme lipidique et du stress oxydant et de déterminer d‘éventuelles corrélations entre le 7β-OHC et les différents mécanismes associés à la pathogenèse de la SEP. Une étude in vitro a été réalisée afin d’évaluer les activités biologiques de trois molécules utilisées dans le traitement de la SEP, le diméthyle fumarate (DMF) et son métabolite le monométhyle fumarate (MMF), et la biotine sur des oligodendrocytes murins 158N, et à déterminer leurs potentialités cytoprotectrices en se focalisant sur leurs capacités à s’opposer à la toxicité du 7β-OHC. Pour cela, des techniques spectrophotométriques, analytiques et de biologies moléculaires ont été utilisées.Nos résultats ont révélé une augmentation du taux de HODE associée à une augmentation du taux plasmatiques d’oxystérols, notamment 7KC et 7β-OHC, ainsi qu’une altération du métabolisme d’acides gras au niveau du LCR et du plasma des patients atteints de SEP. L’étude in vitro a montré que le DMF, le MMF et la biotine présentent des capacités à atténuer les effets délétères du 7β-OHC à savoir; la mort cellulaire par oxiapoptophagie définie par l’association de l’apoptose, l’autophagie et le stress oxydant. De plus, ces molécules corrigent les modifications structurales et le déséquilibre du statut redox caractérisé par une surproduction d’espèces radicalaires d’oxygène, une activité accrue des principales enzymes anti-oxydantes et une amplification de l'oxydation de macromolécules induites par le 7β-OHC. Elles atténuent également les dysfonctionnements mitochondriaux et péroxysomaux, les altérations de l’expression de protéines de myéline ainsi que le désordre du profil lipidique, induits par le 7β-OHC.Notre étude apporte des arguments en faveurs de la capacité du DMF, du MMF et de la biotine, à atténuer les phénomènes majeurs associés à la mort des oligodendrocytes qui pourraient contribuer à la démyélinisation. Ceci renforce l'intérêt porté à ces molécules pour le traitement des maladies neurodégénératives incluant la SEP. / Oxidative stress, mitochondrial dysfunction, and alterations in lipid metabolism are a common denominator of neurodegenerative diseases (MN), such as multiple sclerosis (MS). Under oxidative stress conditions, excess cholesterol is removed by oxidation, producing oxysterols. In humans, the 7β-hydroxycholesterol (7β-OHC) is often found at increased levels in the cerebrospinal fluid (CSF) and/or plasma of patients with MN, including MS.In this context, a preliminary clinical study on CSF and plasma of patients with RR MS was carried out to search for lipid metabolism and oxidative stress biomarkers and to determine the possible correlations between 7β-OHC and the different mechanisms associated with the MS pathogenesis. An in vitro study was conducted to evaluate the biological activities of three molecules used in the treatment of MS, dimethyl fumarate (DMF) and its major metabolite, monomethyl fumarate (MMF), and biotin on 158N murine oligodendrocytes, and to determine their cytoprotective potentialities by focusing on their ability to oppose the toxicity of 7β-OHC. For this, spectrophotometric, analytical and molecular biology techniques were used.Our results have shown an increased level of HODE associated with an enhancement of oxysterol levels in the plasma, notably 7KC and 7β-OHC, as well an alteration in fatty acid metabolism in the CSF and plasma of patients with SEP. The in vitro study revealed that DMF, MMF, and biotin can counteract the deleterious effects of 7β-OHC namely; cell death by oxiapoptophagy defined by the association of apoptosis, autophagy and oxidative stress. Moreover, these molecules correct the structural modifications and the disequilibrium of the redox status characterized by an overproduction of radical oxygen species, an increased activity of the principal antioxidant enzymes and an amplification of the macromolecules oxidation induced by the 7β- OHC. They also attenuate the mitochondrial and peroxisomal dysfunctions, the alterations of myelin protein expression as well as the lipid profile disorder induced by 7β-OHC.Our study provides arguments in favor of the ability of DMF, MMF, and biotin, to attenuate the major events associated with the death of oligodendrocytes which could contribute to demyelination. This reinforces the interest in these molecules for the treatment of neurodegenerative diseases including MS.

The aim of this report is to present a basis for development of an alternative quality control method for a part of the cancer monitoring technique DiviTum® by Biovica. The methods recommended are direct implementation of bromodeoxyuridine (BrdUTP), as well as biotin and digoxigenin systems. All methods show great potential since they do not require acquisition of new instruments or a change of microtiter plate. Further, all have specificity for the poly-A templates, include fewer steps, and are relatively cheap which aligns with the delimitations of the project. Additional methods are discussed to gain an overview of possible quality controls. However, these methods do not align with all the delimitations and are thus not recommended. An ethical analysis was conducted in which issues related to the suggested quality control methods were discussed. These were for example the potential for reusability of materials, and some ethical considerations that arise when handling biological samples from patients or donors. The results obtained are based on a literature study as well as a complementary interview.

Neurons depend upon neurotransmitter release through regulated exocytosis to accomplish the immense processing performed within the central nervous system. The SNARE hypothesis points to a family of proteins that are thought to enable the membrane fusion that leads to exocytosis. The secondary structure of SNAP-25 is unique among SNARE proteins in that it has two alpha helical SNARE motifs and a cysteine rich (C85, C88, C90, C92) membrane interacting region but notransmembrane domain. The cysteines may be modified by palmitoylation or oxidation but the role of these modifications in vivo is not well understood. Our goal is to elucidate possible regulatory roles of SNAP-25 that relate to its unique structure and these reversible modifications. However, the study of SNAP-25 in reconstituted systems is hampered by a lack of readily available palmitoylated SNAP-25. A method for in vitro palmitoylation of SNAP-25 by HIP14, a neuronal acyltransferase, is described along with the application of a biotinylation streptavidin assay to verify palmitoylation. Palmitoylation increases the extent to which SNAP-25 interacts with lipids as observed with an environment sensitive trpytophan fluorescence assay. Palmitoylation also alters the phase transition of DPPC lipids differently than unpalmitoylated SNAP-25.This effect on the membrane may influence fusion events. Oxidation of the cysteine residues may be responsible for the sensitivity of SNAP-25 to reactive oxygen species. Our data suggests that, when oxidized, SNAP-25 does not interact with membranes to the same extent as palmitoylated SNAP-25. This may provide a mechanism for reducing exocytosis during oxidative stress. Also, oxidized SNAP-25 is not susceptible to Botulinum Neurotoxin E. The effects of oxidation and palmitoylation on the protein interactions of SNAP-25 may shed light on its role in the SNARE complex and membrane fusion.

No description available.

The Streptomyces avidinii protein streptavidin binds the small molecule biotin (vitamin H / B₇) with extraordinary stability, resulting in the streptavidin-biotin interaction being one of the strongest non-covalent interactions known in nature (K_d ~ 10^-14 M). The stable and rapid biotin-binding, together with high resistance to heat, pH and proteolysis, has given streptavidin huge utility, both in vivo and in vitro. Accordingly, streptavidin has become a widely used tool in many different biotechnological applications. Streptavidin has also been the subject of extensive research efforts to glean insights into this paradigm for a high-affinity interaction, with over 200 mutants of the protein reported to date. Despite the high stability of the streptavidin-biotin interaction, it can and does fail under certain experimental conditions. For example, streptavidin-biotin dissociation is accelerated by an increased temperature or lower pH (conditions often encountered in cellular imaging experiments), and by mechanical stress, such as the shear force arising from fluid flow (encountered when streptavidin is used as a molecular anchor in biosensor chips and arrays). This study details efforts made at increasing further the utility of streptavidin, by increasing the stability of biotin and biotin-conjugate binding. A rational site-directed mutagenesis approach was used to create 27 mutants, with eight of these mutants possessing higher-stability biotin-binding. The most stable biotin-binding mutant was named traptavidin and was extensively characterised. Kinetic characterisation revealed traptavidin had a decreased dissociation rate from biotin and biotin-conjugates when compared to wildtype streptavidin, at both neutral pH and pH 5. Atomic force microscopy and molecular motor displacement assays revealed the traptavidin-biotin interaction possessed higher mechanical stability than the streptavidin-biotin interaction. Cellular imaging experiments revealed the non-specific cell binding properties of streptavidin were unchanged in traptavidin. X-ray crystallography was also used to generate structures of both apo- and biotinbound traptavidin at 1.5 Å resolution. The structures were analysed in detail and compared to the published structures of streptavidin, revealing the characteristics of traptavidin arose from the mutations stabilising a flexible loop over the biotin-binding pocket, as well as reducing the conformational change on biotin-binding to traptavidin. Traptavidin has the potential to replace streptavidin in many of its diverse applications, as well as providing an insight into the nature of ultra-stable noncovalent interactions.

Despite recent advances in stem cell biology, immunotherapy and transplantation, substantial barriers still exist in the large-scale specific separation of a discrete population of human therapeutic cells from a cell suspension. The ideal purification technique should combine high cell purity, yield and function, with fast processing and affordability. Currently, fluorescence-activated cell sorting with flow cytometry (FACS) and magnetic activated cell sorting (MACS®) are the most used methods for cell separation and purification and have been employed extensively in molecular biology, diagnostic and cell sorting applications, because they are considered to be gentle, fast and scalable. However, these methods have several key disadvantages; they are invariably expensive, yield low log cell reduction (LCR) rates, and suffer from drawbacks when applied to niche cell populations, such as those requiring multiple tandem separation steps and/or involving combined positive and negative cell selection steps. To address this challenge, a new cell affinity selection system was developed. The selectivity is based on the reversible monomeric avidin biotin interaction and it is primary designed for positive selection. The initial studies were performed on flat, nonporous, glass coverslips and the technology was then successfully transferred on high grade smooth non-porous glass beads (with a diameter of 79.12 to 118.59 μm). The multi-step layer-by-layer deposition procedure culminating in dextran-coated supports bearing monomeric avidin was rigorously characterized and subsequently employed in packed bed chromatography experiments with human erythrocytes isolated from cord blood and B lymphocytes from cell lines. The developed affinity selection platform was highly selective, efficient and, most importantly, resulted in high yields, cell purity and viability comparable with MACS® technology. Additionally scale up is possible and could be easily transferred to another chromatographic matrix with the appropriate structure.

L’objectif de cette thèse a été de synthétiser, caractériser et évaluer l’efficacité de photosensibilisateurs (PS) activables par excitation mono- et bi-photonique pour la thérapie photodynamique (PDT). Les PSs sont des dérivés de porphyrines qui ont été associés à différents composés incluants des sondes d’imagerie, des vecteurs, ou des nanoparticules, dans le but d’améliorer l’efficacité du traitement. Un PS a ainsi été relié à deux complexes de Gd(III) pour former un agent moléculaire théranostique afin de pouvoir suivre l’évolution de la PDT par imagerie par résonance magnétique (IRM). Cet agent théranostique conserve les bonnes propriétés photophysiques du PS et sa relaxivité remarquable permet l’imagerie à des concentrations plus faibles que les agents de contraste commerciaux. L’association d’un PS avec un vecteur, l’acide folique ou la biotine, a permis d’augmenter la sélectivité envers les cellules cancéreuses. L’augmentation de la phototoxicité envers des cellules HeLa pour ces deux PSs par rapport au PS non vectorisé montre le potentiel de ces deux PSs vectorisés pour la PDT par excitation mono et bi-photonique. Enfin, plusieurs PS ont été liés ou incorporés dans des cubosomes pour améliorer leur biodistribution et leurs phototoxicités ont été étudiées. Ces études montrent pour la première fois la possibilité d’utiliser des cubosomes comme formulation pour les PSs. / The aim of this thesis was to synthesize, characterize and evaluate the efficiency of new photosensitizers (PS) for Photodynamic Therapy (PDT) activated by a one- or two-photon absorption. Theses PSs are composed of π-extended porphyrins linked to imaging probes, targeting moieties or nanoparticles to improve the treatment efficiency. A PS was linked to two Gd(III) complexes to form a molecular theranostic agent allowing the monitoring of the PDT outcome by Magnetic Resonance Imaging (MRI). This molecular theranostic agent retains the good photophysical properties of the PS and its high relaxivity allows the imaging at a lower concentration than the commercial contrast agents. In order to increase the selectivity towards cancer cells a PS was linked to folic acid or biotin, two targeting compounds. The increase of the phototoxicity towards HeLa cells of these two targeted PSs compared to a non-targeted PS reveals the potential for one- and two-photon PDT. Finally, several PSs have been linked to cubosomes to increase the biodistribution and their phototoxicity has been investigated. This study shows for the first time that cubosomes can be used as a formulation for PSs.

Após a descrição da Urocortina 1, um neuropeptídeo encontrado principalmente no núcleo de Edinger-Westphal e no núcleo lateral superior da oliva (LSO), atentou-se para a ausência do conhecimento das projeções de ambos os núcleos. Nós pretendemos contribuir para o conhecimento das projeções ascendentes e descendentes do LSO, usando um traçador neuronal anterógrado. Nós utilizamos o Biotin-Dextran-Amine (BDA) injetado no LSO de ratos, 15 a 20 dias depois os ratos eram perfundidos, os encéfalos e medulas foram seccionados e tratados histoquimicamente. Nós encontramos que existem 4 vias eferentes do LSO, tanto ascendentes como descendentes no sistema nervoso central, como segue: duas vias ascendentes, uma ipsilateral à injeção, a mais proeminente e a via contralateral que é menos densa; duas vias descendentes, uma ipsilateral muito menos evidente, e a contralateral que é moderada. Seguindo a via ascendente ipsilateral, nós encontramos as seguintes estruturas bem marcadas com BDA: o próprio LSO, núcleo do corpo trapezóide, o lemnisco lateral e seus núcleos, colículos inferior e superior e os seguintes núcleos talâmicos: suprageniculado, geniculado medial, partes dorsal e medial e córtex somatosensorial primário. Seguindo a via descendente contralateral nós encontramos as seguintes estruturas: o LSO ipsi e contralateral, o núcleo do corpo trapezóide, núcleo coclear ventral, parte anterior, núcleo coclear dorsal, núcleo coclear ventral, parte posterior e VIII nervo. Os dados que nós encontramos neste trabalho sugerem que as vis do LSO podem se estender até o córtex somatosensorial no prosencéfalo e o complexo de núcleos cocleares no tronco, enviando colaterais para os principais núcleos relacionados a via auditiva, provavelmente contribuindo para a localização da fonte sonora, em acordo com a anatomia desta informação sensorial específica. / After the discovery of the Urocortin-1, a neuropeptide found mainly in the Edinger-Westphal nucleus and in the lateral superior olive nucleus (LSO), the attention was caught about the lack of known projections of both nuclei. We intended to contribute to the knowledge of both ascending and descending projections of the LSO, using a neuronal anterograde tracer. In order to do that we use the Biotin-Dextran-Amine (BDA) injected in the LSO of rats, fifteen to twenty days later the rats were perfused, the brains and spinal cords were cut and the sections treated histochemically. We have found that there are four pathways leaving the LSO either ascending or descending in the central nervous system, as following: two ascending pathways, one ipsilateral to the injection, the most proeminent one and the contralateral pathway that is less dense; two descending pathways, one ipsilateral, much less evident, and the contralateral that is very moderate. Tracking the ipsilateral ascending pathway we have found the following structures well labeled with BDA: the LSO itself, nucleus of the trapezoid body, the lateral lemniscus and its nuclei, inferior and superior colliculus, the following thalamic nuclei: suprageniculate, medial geniculate, dorsal and medial parts and the primary somatosensory cortex. Tracking the contralateral descending pathway we have found the following structures: the LSO ipsi and contralateral; the nucleus of the trapezoid body; ventral cochlear nucleus, anterior part; dorsal cochlear nucleus; ventral cochlear nucleus, posterior part and, the eight nerve. The data we have found in this work suggests that the pathways from the LSO could reach as far as the somatosensory cortex in the prosencephalon and the cochlear complex nuclei in the brainstem, sending collaterals to the main nuclei related to the auditory pathway, probably contributing to the localization of the sound source, due to the anatomy of this specific sensory information.

O neuropeptídeo urocortina 1 (UCN 1) tem entre os seus principais locais de expressão o núcleo de Edinger-Westphal (EW) e o núcleo lateral superior da oliva. Após sua descoberta, sugeriu-se que o EW e o núcleo paraventricular do hipotálamo (PVH) possuíssem papéis complementares e opostos na resposta ao estresse, porém, não existem trabalhos que relacionam anatomicamente núcleos hipotalâmicos e o EW. A fim de contribuir para esta área foi proposto o mapeamento das aferências hipotalâmicas do EW, através da injeção de Fluoro-Gold neste núcleo e posterior mapeamento de suas aferências. Os resultados encontrados foram: PVH, área hipotalâmica lateral (LHA) e o núcleo posterior do hipotálamo (PH) e outras regiões do sistema nervoso central. Para controle, o traçador anterógrado Amina Dextrana Biotinilada, foi injetado nos núcleos/áreas hipotalâmicas PVH, LHA e PH sendo encontradas fibras próximas as células urocortinérgicas do EW. Nossos dados mostram um possível envolvimento das células UCN 1 do EW com o controle de funções autonômicas e neuroendócrinas. / The neuropeptide urocortin 1 (UCN 1) has its main sites of expression at the Edinger-Westphal nucleus (EW) and the lateral superior olivary nucleus. After its discovery has suggested that EW and paraventricular nucleus of hypothalamus (PVH) have complementary and opposing roles in the stress response. However, there are no works relating anatomically the hypothalamic nuclei and EW. To contribute to this area we proposed mapping the hypothalamic afferents of the EW. We have used the Fluoro-Gold injected in the EW as a result of we have found retrogradely labeled cells in the following nuclei: PVH, lateral hypothalamic area (LHA), posterior hypothalamic nucleus (PH) and other regions of the central nervous system. For control, the anterograde tracer biotinylated dextran amine was injected into the nuclei/areas hypothalamic PVH, LHA, and PH we have found anterogradely labeled fibers in a very close apposition over urocortinergic cells at EW. Based on these data we are suggesting a involvement of cells with UCN 1 EW control of autonomic and neuroendocrine functions.