Doctor Pharmaceuticae - Dpharm / Among neurological disorders, many of the most devastating disorders are neurodegenerative. Modern research associates excitotoxicity to a variety of neuropathological conditions, suggesting that the neurodegenerative diseases with distinct etiologies may have excitotoxicity as a common pathway. Excitotoxicity occurs through over-stimulation of receptors for excitatory neurotransmitters like the N-methyl-D-aspartate (NMDA) receptors. Due to the relevance of NMDA receptors and excitotoxic processes, the antagonism or modulation of NMDA receptors is used as a therapeutic tool against neurodegenerative diseases. NMDA receptor activity can be modulated by S-nitrosylation and this modulation of NMDA receptor activity can be utilised in the development of neuroprotective drugs.

Neste trabalho foram investigados os mecanismos e o perfil cinético de processos nitrosativos do ponto de vista da nitrosação do indicador 4,5-diamino fluoresceina (DAF2) em células do tipo RAW 264.7. Também foi investigado o papel que ferro lábil (LIP) exerce em tais processos. O estudo cinético mostrou que a nitrosação do DAF2 é dependente de superóxido intracelular e se processa por dois mecanismos distintos denominados nitrosilação oxidativa e nitrosação. Observou-se que o perfil cinético da nitrosaçao do DAF2 sofre uma transição passando de dependente para independente com relação à concentração de NO, quando a concentração de NO se aproxima de 100-110nM. Este perfil está relacionado com a dinâmica de recombinação entre NO e O2¯ que dispara todo o processo de nitrosação do DAF2. No trabalho fica claro que processos nitrosativos que ocorrem pelos mesmos mecanismos podem apresentar perfis cinéticos completamente diferentes dependendo da localização onde ocorre a recombinação entre NO e O2¯. O ponto mais interessante foi a constatação de que quelantes permeáveis à membranas biológicas estimulam a nitrosação do DAF2 intracelular. Este efeito é decorrente da remoção de LIP intracelular que, surpreendementemente, apresenta papel antinitrosativo nas condições experimentais estudadas. O papel incomum antinitrosativo apresentado por LIP é analizado do ponto de vista da reação entre LIP e ONOO¯ que tem como produto nitrito, uma espécie não nitrosante. Estes resultados podem alterar a forma como LIP é visto em processos oxidativos e nitrosativos. / In this work, we investigated the mechanisms and kinetic profiles of nitrosative processes using fluorescent indicator 4,5-diaminofluorescein (DAF2) in RAW 264.7 cells. The labile iron pool (LIP) influence in nitrosative processes was also evaluated. Intracellular DAF2 nitrosation is superoxide dependent and proceeds by two distinct mechanisms: Oxidative nitrosylation and nitrosation. The former mechanism is the most relevant under all experimental conditions tested. Interestingly, the DAF2 nitrosation rate increases linearly with NO concentration of up 100-110 nM but thereafter undergoes a sharp transition and becomes insensitive to NO. This peculiar kinetic behavior has never been reported and it is linked with NO and superoxide recombination dynamics. When NO reaches a concentration capable to outcompete superoxide dismutase for superoxide, the rate of DAF2 nitrosation becomes insensitive to NO. The most striking finding is the LIP´s influence in nitrosative processes. LIP removal by cell membrane permeable metal chelantors increases DAF2 nitrosation rate significantly, suggesting tha LIP can act as an anti-nitrosant species. This increase is probably related with LIP´s direct reaction with peroxynitrite, wich produces non-nitrosant species like nitrite. This controversial LIP´s anti-nitrosative role in cellular systems is rather interesting since it can change the way we understand it´s role in nitrosative and oxidative processes.

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.