A exposição de animais a situações ameaçadoras de natureza inata ou aprendida resulta em exibição de um repertório de comportamentos defensivos espécie-específicos, alterações autonômicas e em inibição da dor, sendo esse conjunto de reações de alta relevância para a sobrevivência de uma espécie. Considerando este contexto, um importante componente da resposta do organismo a situações de emergência é a redução da capacidade de perceber a dor. O processamento de estímulos nociceptivos pode ser modulado no prosencéfalo, na medula espinal, no tronco encefálico e no diencéfalo, por mecanismos envolvendo diferentes neurotransmissores e neuromoduladores. Nos últimos anos, evidências têm demonstrado que o monóxido de carbono (CO), produzido a partir da enzima heme-oxigenase estimula a formação de guanosina 3, 5- monofosfato cíclico (GMPc), participando como neuromodulador de vários processos fisiológicos. Dentro deste contexto, mostrou-se que a via HO-CO-GMPc está envolvida na modulação periférica e espinal da dor inflamatória, bem como na modulação do estresse, porém não há conhecimento da participação desta via na modulação de estímulo doloroso agudo, bem como da antinocicepção induzida pelo estresse. Assim, este trabalho teve como objetivo avaliar o envolvimento da via HO-CO-GMPc na nocicepção e na antinocicepção induzida pelo estresse agudo em ratos, avaliada pelo índice de analgesia no teste de retirada da cauda (IARC). Nossos resultados demonstraram que a ativação da via HO-CO-GMPc por meio da administração ICV de heme-lisinato (substrato) tem efeito antinociceptivo, sendo este efeito dependente da atividade GMPc, desde que o pré-tratamento com inibidor da guanilase ciclase solúvel (GCs), ODQ, bloqueou o aumento do IARC. Ainda, esta modulação ocorre de maneira fásica e não tônica, pois o tratamento isolado ICV com o inibidor da HO, ZnDBPG ou com o inibidor da GCs, ODQ, não alterou o IARC. A antinocicepção induzida pelo estresse agudo (restrição física por 120 min) não é dependente da via HO-CO-GMPc, desde que o tratamento com o ZnDBPG, nem com o heme-lisinato alteraram o IARC. No entanto, esta antinocicepção é dependente da atividade do GMPc, pois o pré-tratamento com ODQ bloqueou o aumento do IARC. / The exposure of animals to threatening situations of innate or learned nature results in exhibition of a repertoire of species-specific defensive behaviors, autonomic alterations and pain inhibition. This group of reactions has high relevance for the survival of species. In this context, an important component of the response of the organism in the emergency situations is the reduction of the capacity to perceive pain. The processing of nociceptive stimulus can be modulated in forebrain, in spinal, and in midbrain, for mechanisms involving different neurotransmitters and neuromodulators. Recently, evidence has demonstrated that carbon monoxide gas (CO), produced from the enzyme heme oxygenase (HO), stimulate the formation of 3\', 5\' - cyclic guanosine monophosphate (cGMP), and this molecule has participated as neuromodulator in some physiological processes. In this way, it has shown that the HO-CO-cGMP pathway is involved in the peripheral and spinal modulation of inflammatory pain, as well as in the modulation of the stress. However, the involvement of this pathway in the modulation of acute painful stimulus, as well as in the antinociception induced by stress isn´t clarified. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in nociception and in antinociception induced by acute stress in rats, by means the of analgesia index in the tail flick test (AITF). Our results demonstrated that the activation of the HO-CO-cGMP pathway by means of heme-lysinate ICV administration has antinociceptive effect. Again, the increase of the AITF was dependent of the cGMP activity, since that the pretreatment with inhibitor of soluble guanylase cyclase (sGC), ODQ, blocked the antinociceptive effect. This modulation occurs in fasic and not tonic manner, because per se ICV treatment with inhibitor of the HO, ZnDBPG or with inhibitor of the sGC, ODQ did not modify the AITF. The antinociception induced by acute stress (physical restriction during 120 min) is not dependent of the HO-CO-cGMP pathway, since that neither the treatment with the ZnDBPG, nor with the heme-lysinate had modified the AITF. However, this antinociception is dependent of the activity of the cGMP, because the pretreatment with ODQ blocked the increase of the AITF induced by acute stress.

No description available.

<p>Exchange proteins directly activated by cAMP (EPAC) are guanine nucleotide exchange factors for the small GTPases, Rap1 and Rap2. The central regulatory module of EPAC is a cAMP binding domain (CBD), which in the absence of cAMP provides auto-inhibition of the catalytic guanine nucleotide exchange activity. Binding of the allosteric effector, cAMP, removes the auto-inhibition exerted by the CBD of EPAC. Herein, we investigate through NMR spectroscopy the structural and dynamical basis of auto-inhibition and cAMP-dependent allosteric activation in the CBD of EPAC. Specifically, the work described in this dissertation proposes novel methods that utilize NMR chemical shifts to define the network of residues that mediates long-range intra-molecular signalling, <em>i.e.</em> the chemical shift covariance analysis (CHESCA) and the chemical shift projection analysis (CHESPA). Using CHESCA as explained in Chapter 2, we identified an allosteric network that bridges the sites of cAMP-binding and cAMP-dependent structural changes to those of cAMP-dependent dynamical changes, which are critical for the release of auto-inhibition. The CHESCA results therefore rationalize how cAMP leads to activation through modulation of both structure and dynamics. In order to dissect the determinants of auto-inhibition in the absence of cAMP, several mutations along the signaling pathways identified by CHESCA were implemented and their effect on the auto-inhibitory conformational equilibrium of the apo-CBD was assessed through CHESPA, as outlined in Chapters 3 and 4. Overall, we have shown how CHESCA and CHESPA provide unprecedented insight into the allosteric networks underlying auto-inhibition and cAMP dependent activation in the CBD of EPAC. In addition, the methods employed here to map EPAC allostery are likely to be generally applicable to other systems.</p> / Doctor of Philosophy (PhD)

Genetisch kodierte Photorezeptoren in Modellorganismen begründen die Optogenetik. Sie ermöglicht die nicht-invasive, reversible und räumlich-zeitlich präzise Perturbation von zellulären und physiologischen Signalprozessen durch Licht. Natürliche photoaktivierte Adenylylzyklasen (PACs) steigern die intrazelluläre Konzentration des Botenstoffs zyklischen Adenosinmonophosphats (cAMP) durch Blaulicht. Damit erlauben sie die optogenetische Analyse von cAMP-abhängigen Signalwegen. Diese Arbeit komplementiert PACs durch die synthetische rotlichtaktivierte Phosphodiesterase LAPD zur Degradation von cAMP und zyklischem Guanosinmonophosphat (cGMP). LAPD ist eine Chimäre aus dem photosensorischen Modul von Deinococcus radiodurans Bakteriophytochrom (DrBPhy) und der Effektordomäne der cAMP/cGMP-spezifischen H. sapiens Phosphodiesterase 2A (HsPDE2A). Die Fusionsstelle wurde von den helikalen Linkern zwischen Sensor- und Effektormodulen durch strukturelle Überlagerung abgeleitet. LAPD inkorporierte den Chromophor Biliverdin (BV) nach Expression in E. coli und Reinigung vollständig und entsprach spektral und photochemisch dem Wildtyp-DrBPhy. Durch Bestrahlung mit Rot- und Fernrotlicht (R bzw. FR) wurde LAPD in die metastabilen photochemischen Zustände Pfr (fernrot) bzw. Pr (rot) umgewandelt. Vollständig aktivierte LAPD katalysierte die Hydrolyse von cGMP und cAMP in derselben Größenordnung wie Wildtyp-HsPDE2A. LAPD degradierte cGMP und cAMP bei 6- bzw. 4-facher Steigerung von vmax unter R im Vergleich zu dunkeladaptiertem Enzym. Die Aktivität von R-adaptierter LAPD wurde durch FR reduziert. Die enzymatische Aktivität und Lichtregulation von LAPD-Linkervarianten waren abhängig von der Linkerlänge. LAPD degradierte lichtabhängig cGMP in einer PDE-Reporterzelle. Dabei genügte die endogene BV-Konzentration der Säugerzelle zur Sättigung des Lichteffekts. / Genetically encoded photoreceptors in model organisms establish optogenetics. It enables non-invasive, reversible, and spatio-temporally precise perturbation of cellular and physiological signalling by light. Natural photoactivated adenylate cyclases (PACs) increase the intracellular concentration of the second messenger cyclic adenosine monophosphate (cAMP) under blue light. Hence, PACs allow the optogenetic analysis of cAMP-dependent signalling. This work complements PACs with the synthetic red-light-activated phosphodiesterase LAPD for degradation of cAMP and cyclic guanosine monophosphate (cGMP). LAPD is a chimera made up of the photosensory module of Deinococcus radiodurans bacteriophytochrome (DrBPhy) and the effector domain of cAMP/cGMP-specific H. sapiens Phosphodiesterase 2A (HsPDE2A). The fusion site was derived from the helical linkers between sensor and effector modules via structural superposition. LAPD incorporated the chromophor biliverdin (BV) after expression in E. coli and purification quantitatively, and spectrally and photochemically resembled the wildtype DrBPhy. Upon irradiation with red and far-red light (R and FR, resp.), LAPD was converted to the metastable photochemical states Pfr (far-red) and Pr (red), respectively. Fully activated LAPD catalized the hydrolysis of cGMP and cAMP with rates similar to wildtype HsPDE2A. LAPD degraded cGMP and cAMP with 6- and 4-fold increase of vmax under R, respectively, as compared to the dark state. The activity of R-adapted LAPD was reduced upon irradiation with FR. Enzymatic activity and light regulation of LAPD linker variants depended on the linker length. LAPD light-dependently degraded cGMP in a PDE reporter cell line. Endogenous BV concentrations were sufficient to saturate the light effect in the mammalian cell, which enables a true optogenetic approach.

La découverte du système des peptides natriurétiques (NP), au début des années 80, fut une découverte majeure qui révéla le rôle endocrinien du cœur. Les connaissances sur la relaxation vasculaire, la diurèse et la natriurèse provoquées par ce système ont évolué vers un niveau de complexité insoupçonné à cette époque. Nous savons à présent que les NP sont impliqués dans plusieurs autres mécanismes dont la prolifération cellulaire, l’apoptose, l’inhibition du système rénine-angiotensine-aldostérone (RAAS) et le métabolisme des adipocytes. Le métabolisme des lipides est maintenant devenu une cible de choix dans la lutte contre l’obésité. Cette condition aux proportions pandémiques est un facteur de risque majeur dans l’apparition de l’hypertension et du syndrome métabolique (MetS). La compréhension des mécanismes et des défauts de la voie des NP pourrait avoir un impact positif sur le contrôle du MetS et de l’hypertension. L’expression du récepteur des peptides natriuretiques de type 1 (NPR1/GCA) est contrôlée par plusieurs agents incluant son propre ligand, le peptide natriurétique de l’oreillette (ANP). La découverte d’une boucle de retro-inhibition, dans les années 90, a été un événement majeur dans le domaine des NP. En effet, suite à une stimulation à l’ANP, le NPR1/GCA peut inhiber l’activité transcriptionnelle de son propre gène par un mécanisme dépendant du cGMP. Notre groupe a identifié un élément cis-régulateur responsable de cette sensibilité au cGMP et mon projet consistait à identifier la ou les protéine(s) liant cet élément de réponse au cGMP (cGMP-RE). Nous avons identifié un clone liant le cGMP-RE en utilisant la technique du simple hybride chez la levure et une banque d’ADN complémentaire (ADNc) de rein humain. Ce clone provient d’un ADNc de 1083-bp dont le gène est localisé sur le chromosome 1 humain (1p33.36) et codant pour une protéine dont la fonction était inconnue jusqu’ici. Nous avons nommé cette nouvelle protéine GREBP en raison de sa fonction de cGMP Response Element Binding Protein. Des essais de liaison à l’ADN ont montré que cette protéine possède une affinité 18 fois plus élevée pour le cGMP-RE que le contrôle, tandis que des expériences de retard sur gel (EMSA) ont confirmé la spécificité des interactions protéine-ADN. De plus, l’immuno-précipitation de la chromatine (ChIP) a prouvé que GREBP lie le cGMP-RE dans des conditions physiologiques. La liaison de GREBP au cGMP-RE inhibe l’expression du gène rapporteur luciférase sous contrôle du promoteur de npr1/gca. L’inhibition de GREBP à l’aide d’ARN interférant active le promoteur de npr1/gca. Dans les cellules NCI-H295R, l’ANP stimule l’expression de grebp de 60% après seulement 3 heures et inhibe l’expression de npr1/gca de 30%. GREBP est une protéine nucléaire surtout exprimée dans le cœur et ayant le facteur eIF3F comme partenaire. Les variations nucléotidiques du gène sont plus fréquentes chez les patients hypertendus que chez des patients normotendus ou hypertendus souffrant de MetS. Nous rapportons ici l’existence d’un gène spécifique à l’humain qui agit comme répresseur transcriptionnel de npr1/gca et potentiellement impliqué dans le développement de l’hypertension. / The natriuretic peptide (NP) system was a milestone discovery that revealed the endocrine role of the heart for the first time in the early 1980s. From its vasodilatory, natriuretic and diuretic actions, knowledge about this system has evolved to a degree of complexity unsuspected at that time. Now, through cGMP generation, NPs are involved in several other mechanisms, such as cell proliferation, apoptosis, renin-angiotensine-aldosterone system (RAAS) inhibition, and fat cell function. The latter point is of growing interest in lipid metabolism and has become an important issue in the fight against obesity. This pandemic condition is one of the main risk factors leading to hypertension development and metabolic syndrome (MetS) progression. Thus, understanding, at least in part, the lipid mobilization pathways controlled by NPs could have a positive impact in MetS management. As with hypertension, identifying defects in signaling pathways will certainly help to identify mechanisms implicated in lost sensitivity of the NP system. Natriuretic peptide receptor 1 (npr1/gca) expression is controlled by several agents including its own ligand, the atrial natriuretic peptide (ANP). A major finding in NPs field occured in the mid-90s when a mechanism involving a retro-inhibition loop was described. Indeed, after ANP stimulation, NPR1/GCA down-regulates the transcriptional activity of its gene via a cGMP-dependent mechanism. Since our group previously identified a cis-acting element responsible for this cGMP sensitivity, I proceeded to explore novel putative protein binding to the cGMP-response element (cGMP-RE). Using the yeast-one-hybrid technique with a human kidney cDNA library, we identified a strongly positive clone able to bind cGMP-RE. The clone was derived from a 1083-bp long cDNA of a gene of yet unknown function localized on human chromosome 1 (1p33.36). We named this new protein GREBP for cGMP-Response Element-Binding Protein. DNA-binding assays showed 18-fold higher cGMP-RE-binding capacity than the controls while electromobility shift assay (EMSA) indicated a specific binding for the cGMP-RE and chromatin immuno-precipitation (ChIP) confirmed the binding of GREBP to the element under physiological conditions. By acting on cGMP-RE, GREBP inhibited the activity of a luciferase-coupled NPR1 promoter construct. In H295R cells, ANP heightened GREBP expression by 60% after just 3 hours of treatment while inhibiting npr1/gca expression by 30%. Silencing GREBP with specific small interfering RNA increased the activity of the luciferase-coupled NPR1/GCA promoter and NPR1/GCA mRNA levels. GREBP is a nuclear protein mainly expressed in the heart and has the eIF3F factor as partner. Its nucleotide variations are more frequent in non-obese hypertensive patients than normotensive subjects or hypertensive patients suffering from MetS. We report here the existence of a human specific gene acting as a transcriptional repressor of npr1/gca gene that could be implicated in hypertension development.

La découverte du système des peptides natriurétiques (NP), au début des années 80, fut une découverte majeure qui révéla le rôle endocrinien du cœur. Les connaissances sur la relaxation vasculaire, la diurèse et la natriurèse provoquées par ce système ont évolué vers un niveau de complexité insoupçonné à cette époque. Nous savons à présent que les NP sont impliqués dans plusieurs autres mécanismes dont la prolifération cellulaire, l’apoptose, l’inhibition du système rénine-angiotensine-aldostérone (RAAS) et le métabolisme des adipocytes. Le métabolisme des lipides est maintenant devenu une cible de choix dans la lutte contre l’obésité. Cette condition aux proportions pandémiques est un facteur de risque majeur dans l’apparition de l’hypertension et du syndrome métabolique (MetS). La compréhension des mécanismes et des défauts de la voie des NP pourrait avoir un impact positif sur le contrôle du MetS et de l’hypertension. L’expression du récepteur des peptides natriuretiques de type 1 (NPR1/GCA) est contrôlée par plusieurs agents incluant son propre ligand, le peptide natriurétique de l’oreillette (ANP). La découverte d’une boucle de retro-inhibition, dans les années 90, a été un événement majeur dans le domaine des NP. En effet, suite à une stimulation à l’ANP, le NPR1/GCA peut inhiber l’activité transcriptionnelle de son propre gène par un mécanisme dépendant du cGMP. Notre groupe a identifié un élément cis-régulateur responsable de cette sensibilité au cGMP et mon projet consistait à identifier la ou les protéine(s) liant cet élément de réponse au cGMP (cGMP-RE). Nous avons identifié un clone liant le cGMP-RE en utilisant la technique du simple hybride chez la levure et une banque d’ADN complémentaire (ADNc) de rein humain. Ce clone provient d’un ADNc de 1083-bp dont le gène est localisé sur le chromosome 1 humain (1p33.36) et codant pour une protéine dont la fonction était inconnue jusqu’ici. Nous avons nommé cette nouvelle protéine GREBP en raison de sa fonction de cGMP Response Element Binding Protein. Des essais de liaison à l’ADN ont montré que cette protéine possède une affinité 18 fois plus élevée pour le cGMP-RE que le contrôle, tandis que des expériences de retard sur gel (EMSA) ont confirmé la spécificité des interactions protéine-ADN. De plus, l’immuno-précipitation de la chromatine (ChIP) a prouvé que GREBP lie le cGMP-RE dans des conditions physiologiques. La liaison de GREBP au cGMP-RE inhibe l’expression du gène rapporteur luciférase sous contrôle du promoteur de npr1/gca. L’inhibition de GREBP à l’aide d’ARN interférant active le promoteur de npr1/gca. Dans les cellules NCI-H295R, l’ANP stimule l’expression de grebp de 60% après seulement 3 heures et inhibe l’expression de npr1/gca de 30%. GREBP est une protéine nucléaire surtout exprimée dans le cœur et ayant le facteur eIF3F comme partenaire. Les variations nucléotidiques du gène sont plus fréquentes chez les patients hypertendus que chez des patients normotendus ou hypertendus souffrant de MetS. Nous rapportons ici l’existence d’un gène spécifique à l’humain qui agit comme répresseur transcriptionnel de npr1/gca et potentiellement impliqué dans le développement de l’hypertension. / The natriuretic peptide (NP) system was a milestone discovery that revealed the endocrine role of the heart for the first time in the early 1980s. From its vasodilatory, natriuretic and diuretic actions, knowledge about this system has evolved to a degree of complexity unsuspected at that time. Now, through cGMP generation, NPs are involved in several other mechanisms, such as cell proliferation, apoptosis, renin-angiotensine-aldosterone system (RAAS) inhibition, and fat cell function. The latter point is of growing interest in lipid metabolism and has become an important issue in the fight against obesity. This pandemic condition is one of the main risk factors leading to hypertension development and metabolic syndrome (MetS) progression. Thus, understanding, at least in part, the lipid mobilization pathways controlled by NPs could have a positive impact in MetS management. As with hypertension, identifying defects in signaling pathways will certainly help to identify mechanisms implicated in lost sensitivity of the NP system. Natriuretic peptide receptor 1 (npr1/gca) expression is controlled by several agents including its own ligand, the atrial natriuretic peptide (ANP). A major finding in NPs field occured in the mid-90s when a mechanism involving a retro-inhibition loop was described. Indeed, after ANP stimulation, NPR1/GCA down-regulates the transcriptional activity of its gene via a cGMP-dependent mechanism. Since our group previously identified a cis-acting element responsible for this cGMP sensitivity, I proceeded to explore novel putative protein binding to the cGMP-response element (cGMP-RE). Using the yeast-one-hybrid technique with a human kidney cDNA library, we identified a strongly positive clone able to bind cGMP-RE. The clone was derived from a 1083-bp long cDNA of a gene of yet unknown function localized on human chromosome 1 (1p33.36). We named this new protein GREBP for cGMP-Response Element-Binding Protein. DNA-binding assays showed 18-fold higher cGMP-RE-binding capacity than the controls while electromobility shift assay (EMSA) indicated a specific binding for the cGMP-RE and chromatin immuno-precipitation (ChIP) confirmed the binding of GREBP to the element under physiological conditions. By acting on cGMP-RE, GREBP inhibited the activity of a luciferase-coupled NPR1 promoter construct. In H295R cells, ANP heightened GREBP expression by 60% after just 3 hours of treatment while inhibiting npr1/gca expression by 30%. Silencing GREBP with specific small interfering RNA increased the activity of the luciferase-coupled NPR1/GCA promoter and NPR1/GCA mRNA levels. GREBP is a nuclear protein mainly expressed in the heart and has the eIF3F factor as partner. Its nucleotide variations are more frequent in non-obese hypertensive patients than normotensive subjects or hypertensive patients suffering from MetS. We report here the existence of a human specific gene acting as a transcriptional repressor of npr1/gca gene that could be implicated in hypertension development.

The mammalian spinal cord contains the neuronal circuitry necessary to generate rhythmic locomotor activity in the absence of inputs from the higher brain centre or sensory system. This circuitry is regulated by local neuromodulatory inputs, which can adjust the strength and timing of locomotor output. The free radical gas nitric oxide has been shown to act as an important neuromodulator of spinal circuits, which control locomotion in other vertebrate models such as the tadpole and lamprey. Despite this, the involvement of the NO-mediated soluble guanylate cyclase/cyclic guanosine monophosphate secondary messenger-signalling pathway (NO/sGC/cGMP) in mammalian locomotion has largely been under-investigated. The NADPH diaphorase histochemical reaction was used to identify sources of NO in the lumbar spinal cord. The largest population NADPH diaphorase reactive neurons were located in the dorsal horn, followed by the laminae of the ventral horn, particularly around the central canal (lamina X) and lamina VII. NADPH diaphorase reactive neurons were found along a rostrocaudal gradient between lumbar segments L1 to L5. These results show that that discrete neuronal sources of NO are present in the developing mouse spinal cord, and that these cells increase in number during the developmental period postnatal day P1 – P12. NADPH diaphorase was subsequently used to identify NADPH diaphorase reactive neurons at P12 in the mouse model of ALS using the SODG93A transgenic mouse. Physiological recordings of ventral root output were made to assess the contribution of NO to the regulation induced rhythmic fictive locomotion in the in vitro isolated spinal cord preparation. Exogenous NO inhibits central pattern generator (CPG) output while facilitating and inhibiting motor neuron output at low and high concentrations respectively. Removal of endogenous NO increases CPG output while decreasing motor neuron output and these effects are mediated by cGMP. These data suggest that an endogenous tone of NO is involved in the regulation of fictive locomotion and that this involves the NO/sGC/cGMP pathway. Intracellular recordings from presumed motor neurons and a heterogeneous, unidentified sample of interneurons shows that NO modulates the intrinsic properties of spinal neurons. These data suggest that the net effect of NO appears to be a reduction in motor neuron excitability.

Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.

With rapid expansion of interest in multimedia and distributed computing applications across the Internet increases importance of optimized delivery of group traffic. According to current situation the best practice to achieve this goal is multicasting. This masters thesis summarizes multicasting methods and facts. Also it draws conclusions from practical application of presented information on commercial project.

The aim of the work described in this thesis is to investigate the nature and mechanisms of action of repellent cues for growing axons. In particular I try to resolve the controversy in the literature regarding the need for protein synthesis in the growth cone in response to external guidance cues. My results resolve the conflicting data in the literature on Semaphorin-3A signalling, where differing labs had shown that inhibiting protein synthesis either blocks or has no effect upon repulsion. They demonstrate the presence of at least two independent pathways, protein synthesis-dependent mTOR activation and -independent GSK3? activation. The higher sensitivity of the synthesis-dependent pathway, and its redundancy at higher concentrations where synthesis-independent mechanisms can evoke a full collapse response alone, resolve the apparent conflict. My experiments also demonstrated that Nogo-?20, a domain of Nogo-A, requires local protein synthesis to cause collapse. Unlike Semaphorin-3A, the dependence of collapse upon protein synthesis is concentration-independent and does not involve guanylyl cyclase, but it does share a dependence upon mTOR activity and the synthesis of RhoA, sufficient to cause collapse downstream of Semaphorin-3A. The other axon-repelling domain of Nogo-A, Nogo-66, is partially dependent upon the proteasome instead. It does not share a common pathway with Nogo-?20, except that both are RhoA-dependent. I further attempted to identify the nature of a repulsive activity found in grey matter, ruling out a previously suggested candidate identity. Finally, I examined the phenomenon of nitric oxide-induced growth cone collapse. My experiments revealed that S-nitrosylated glutathione causes growth cone collapse through the activity of protein disulphide isomerase. This mechanism shows only a partial dependence upon soluble guanylyl cyclase, but I argue that it has total dependence upon an S-nitrosylated donor. Coupled with its apparent relation to S-palmitoylation, the reciprocal of S-nitrosylation, I propose that nitric oxide causes collapse by crossing the cell membrane to inhibit S-palmitoylation-determined localisation of proteins. These results reveal some of the many pathways involved in growth cone collapse, whose further characterisation may provide new targets for the treatment of injuries of the central nervous system.