Les microorganismes photosynthétiques suscitent un intérêt biotechnologique important pour la production de dihydrogène. La cyanobactérie Synechocystis sp. PCC 6803 est capable d'initier une photoproduction d'hydrogène catalysée par une hydrogénase [NiFe] bidirectionnelle qui se présente sous la forme d'un complexe pentamérique (HoxEFUYH). Toutefois l'inhibition de cette enzyme par l'oxygène émis par le photosystème II rend cette photoproduction transitoire et constitue un verrou majeur au développement de tels procédés. L'exploitation de ces organismes impose une meilleure compréhension des bases moléculaires associées à la sensibilité de l'hydrogénase envers l'oxygène ainsi que des composantes limitant son activité de production d'H2, ce qui implique la connaissance détaillée des jeux d'interactions avec ses partenaires physiologiques NAD(P)+/NAD(P)H.Diverses substitutions d'acides aminés potentiellement impliqués dans la sensibilité de l'enzyme à l'O2 et situés au cœur du site actif (Ileu64, Leu107, Leu112) de la sous-unité catalytique HoxH ont été réalisées. Les résultats in vitro et in vivo indiquent une sensibilité envers l'O2 moindre chez le mutant I64M, qui présente une diffusion limitée et un biais vers l'activité de production d'H2.L'étude des interactions de mutants de délétion des gènes diaphorase hoxE et hoxF avec les cofacteurs NAD(P) a montré que NAD+/NADH semblent être les partenaires privilégiés de l'hydrogénase pour le transfert d'électrons, tandis que le NADPH a un effet activateur sur l'enzyme.Ces études apportent des éléments importants pour envisager une optimisation ciblée et maîtrisée pour la bioproduction d'H2. / Oxygenic photosynthetic organisms are a matter of great biotechnological interest for the production of dihydrogen using what seem to be infinite resources, water and solar energy. The cyanobacterium Synechocystis sp. PCC 6803 encodes a bidirectional [NiFe] hydrogenase consisting of a pentameric complex (HoxEFUYH) that allows it to carry H2 photoproduction. However, it is a transient process, mainly due to the oxygen sensitivity of hydrogenases, O2 being produced at PSII during photosynthesis. Future exploitation of these organisms in bioprocesses requires a better understanding of the molecular bases of O2 sensitivity of the hydrogenase and of the elements limiting H2 evolution which involves detailed knowledge of the interactions of the enzyme with its physiological partners NAD(P)+/NAD(P)H.Various mutants of the Synechocystis hydrogenase were created by genetic engineering, targeting specific amino acid residues (Ileu64, Leu107, Leu112) in the catalytic subunit HoxH identified as putative critical elements for O2 sensitivity. Results obtained in vitro and in vivo indicate that the substitution I64M slightly improves O2 tolerance and alters gas diffusion kinetics with a bias towards H2 production. Studying the interaction of diaphorase gene-deletion mutants hoxF and hoxE with partners NAD(P) showed that NAD+/NADH are the preferential electron acceptor/donor of the hydrogenase, while NADPH is more efficient for enzyme activation.These studies provide first insights on the determinants of the oxygen sensitivity of the hydrogenase of Synechocystis and its activation, which are critical elements to consider in targeted optimization for bioproduction of H2.

La NADPH oxydase, Nox4, appartient à la famille des Nox qui génèrent les espèces radicalaires de l'oxygène, ROS, en transférant un électron à l'oxygène moléculaire. Malgré sa large distribution dans les tissus, Nox4 est encore mal comprise. Contrairement aux autres Nox, Nox4 est unique par son activité constitutive et sa capacité à former H2O2. Les ROS sont des espèces bactéricides dans les phagocytes et des outils de signalisation dans les cellules non phagocytaires en étant associés à de nombreuses pathologies inflammatoires et du vieillissement. Une étude de la structure en lien avec la fonction de Nox4 permettra de mettre l'accent sur un mécanisme de fonctionnement et sur de nouvelles cibles thérapeutiques. 5 nouveaux anticorps monoclonaux ont été générés contre une construction recombinante tronquée (AA: 206-578) de Nox4. La spécificité de 3 anticorps monoclonaux (8E9, 5F9, 6B11) a été confirmée par western blot dans les cellules HEK293 transfectées et le cortex de rein humain. L'anticorps 8E9 est le seul à permettre un marquage des cellules TRex-Nox4 sans perméabilisation par FACS. L'immunofluorescence confocale a montré que Nox4 est localisée dans la zone périnucléaire et le réticulum endoplasmique. La microscopie TIRF a confirmé sa présence dans la membrane plasmique. Un phénomène intéressant est que 5F9 ne détecte pas Nox4 à la membrane plasmique. L'épitope de 8E9 reconnaît une région sur la dernière boucle E extracellulaire de Nox4 (222H-E241), tandis que les anticorps monoclonaux, 6B11 et 5F9 marquent respectivement les régions 6B11 (389S-P416) et 5F9 (392D-F398). Par ailleurs, seuls 5F9 et 6B11 inhibent l'activité de Nox4, ce qui suggère que les deux régions marquées par ces ACm sont impliquées dans le transfert d'électrons. Une étude ciblée sur la boucle E de Nox4 a permis de montrer que le changement de 2 cystéines modifie la nature des ROS générés par Nox4 avec la production de O2- au lieu de H2O2. O2- est mis en évidence par la formation de peroxynitrite en présence de NO. Par ailleurs l'ACm 8E9 diminue la production de H2O2 dans les cellules COS7 qui expriment Nox4 à la membrane plasmique alors que celle de O2- est augmentée. Des constructions recombinantes de Nox4 (native ou tronquée) ont été générées par induction bactérienne, E.Coli, et par un système de transcription/traduction (RTS). Les protéines correspondantes, solubles, ont été produites à grande échelle et l'activité diaphorase mesurée; cette activité est constitutive. L'étude de la topologie membranaire de Nox4 et p22phox a été abordée en préparant des protéines de fusion avec l'ubiquitine marquée à la GFP. Cette méthode, TDUFA, particulièrement originale, devrait permettre d'appréhender la topologie de l'hétérodimère Nox4/p22phox, actif. / NADPH oxidase, Nox4, belongs to the Nox family which could generate reactive oxygen species by transferring an electron to molecular oxygen. Despite its wide distribution in tissues, Nox4 is still poorly understood. Unlike the other Noxes, Nox4 shows some unique characters: the constitutive activity, H2O2 formation. Nox4 involved ROS has been proposed to be implicated in several pathologies. Thus, to study the structure/function and the regulation of the activity of Nox4 will provide new ideas and new drug targets for the effective prevention and treatment of clinical diseases related with ROS. To know more about Nox4, in this study, 5 novel monoclonal antibodies were raised against a truncated recombinant protein (AA: 206-578) of Nox4. The specificity of 3 mAbs (8E9, 5F9, 6B11) was confirmed by western blot analysis in HEK293 transfected cells and human kidney cortex. In FACS studies, only mAb 8E9 could react with intact tet-induced T-RExTM Nox4 cells. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. An interesting phenomena is that mAb 5F9 failed to detect Nox4 at the plasma membrane. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4 (222H-E241), while mAb 6B11 (389S-P416) and 5F9 (392D-F398) are directed to its cytosolic tail. Cell-free oxidase assays showed a moderate but significant inhibition of constitutive Nox4 activity by mAb 5F9 and 6B11. To study the protein region which is responsible for the unique ability of Nox4 of releasing H2O2 rather than O2-, chimeric proteins and mutants were used. E-loop of Nox4 is 28 amino acid longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or change of the two cysteines in the E-loop switch Nox4 from H2O2 to O2- generation. In the presence of a NO donor, the O2--producing Nox4 mutants, but not widetype Nox4, generated peroxynitrite, excluding artifacts of the detection systems as the apparent origin of O2-. A second approach was used to confirm the responsibility of E-loop for the H2O2 formation. In Cos7 cells, which exhibit some plasma membrane expression of Nox4, addition of the mAb 8E9 decreased H2O2 production but increased O2- formation. Unlike Nox1 or Nox2, the E-loop of Nox4 contains a highly conserved histidine H222. Mutation of H222 also switched Nox4 from H2O2 to O2- formation. The structure of the E-loop might hinder O2- egress and/or provide a source for protons to accelerate dismutation to form H2O2. Two bacterial protein expression approaches (in vitro RTS and bacterial induction) were used to produce Nox4 cytosolic tail for characterizing the electronic transfer property of Nox4. The presence of rare codons (1363AGA AGA CUA1371) and high level of hydrophobicity affects the production of soluble and active recombinant Nox4Aqc and Nox4Bqc. After optimization of the conditions, soluble and active recombinant proteins were obtained by RTS or by bacteria induction. The soluble proteins were produced in large scale, purified onto affinity chromatography and were tested for the diaphorase activity (INT and cytochrome c). Results showed that electronic acceptor cytochrome c gives a higher rate than INT. Nox4Aqc produced a lower specific activity by a cell-based system compared to the protein synthesized in cell-free technology. This activity is not stimulated by the addition of cytosolic factors. A new method, topological determination by ubiquitin fusion assay (TDUFA), was used to investigate the topology of Nox4 and p22phox. ubGFP fusion proteins are used as tools to obtain details of membrane protein topology. This method was first validated by using two membrane proteins with known topology and then should get more topology information of Nox4 and p22phox further.

La NADPH oxydase, Nox4, appartient à la famille des Nox qui génèrent les espèces radicalaires de l'oxygène, ROS, en transférant un électron à l'oxygène moléculaire. Malgré sa large distribution dans les tissus, Nox4 est encore mal comprise. Contrairement aux autres Nox, Nox4 est unique par son activité constitutive et sa capacité à former H2O2. Les ROS sont des espèces bactéricides dans les phagocytes et des outils de signalisation dans les cellules non phagocytaires en étant associés à de nombreuses pathologies inflammatoires et du vieillissement. Une étude de la structure en lien avec la fonction de Nox4 permettra de mettre l'accent sur un mécanisme de fonctionnement et sur de nouvelles cibles thérapeutiques. 5 nouveaux anticorps monoclonaux ont été générés contre une construction recombinante tronquée (AA: 206-578) de Nox4. La spécificité de 3 anticorps monoclonaux (8E9, 5F9, 6B11) a été confirmée par western blot dans les cellules HEK293 transfectées et le cortex de rein humain. L'anticorps 8E9 est le seul à permettre un marquage des cellules TRex-Nox4 sans perméabilisation par FACS. L'immunofluorescence confocale a montré que Nox4 est localisée dans la zone périnucléaire et le réticulum endoplasmique. La microscopie TIRF a confirmé sa présence dans la membrane plasmique. Un phénomène intéressant est que 5F9 ne détecte pas Nox4 à la membrane plasmique. L'épitope de 8E9 reconnaît une région sur la dernière boucle E extracellulaire de Nox4 (222H-E241), tandis que les anticorps monoclonaux, 6B11 et 5F9 marquent respectivement les régions 6B11 (389S-P416) et 5F9 (392D-F398). Par ailleurs, seuls 5F9 et 6B11 inhibent l'activité de Nox4, ce qui suggère que les deux régions marquées par ces ACm sont impliquées dans le transfert d'électrons. Une étude ciblée sur la boucle E de Nox4 a permis de montrer que le changement de 2 cystéines modifie la nature des ROS générés par Nox4 avec la production de O2- au lieu de H2O2. O2- est mis en évidence par la formation de peroxynitrite en présence de NO. Par ailleurs l'ACm 8E9 diminue la production de H2O2 dans les cellules COS7 qui expriment Nox4 à la membrane plasmique alors que celle de O2- est augmentée. Des constructions recombinantes de Nox4 (native ou tronquée) ont été générées par induction bactérienne, E.Coli, et par un système de transcription/traduction (RTS). Les protéines correspondantes, solubles, ont été produites à grande échelle et l'activité diaphorase mesurée; cette activité est constitutive. L'étude de la topologie membranaire de Nox4 et p22phox a été abordée en préparant des protéines de fusion avec l'ubiquitine marquée à la GFP. Cette méthode, TDUFA, particulièrement originale, devrait permettre d'appréhender la topologie de l'hétérodimère Nox4/p22phox, actif.

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.

Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes that microbes cannot do. One of the largest challenges is the high cost and instability of enzymes and cofactors. To overcome this obstacle, strong motivations have driven intensive efforts in discovering, engineering, and producing thermostable enzymes. In this project, ribose-5-phosphate isomerase (RpiB), one of the most important enzymes in the pentose phosphate pathway, was cloned from a thermophile Thermotoga maritima, and heterologously expressed in Escherichia coli, purified and characterized. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 80°C and pH 6.5-8.0. It had a half lifetime of 71 h at 60°C, resulting in its turn-over number of more than 2 x108 mol of product per mol of enzyme. Another two thermostable enzymes glucose-6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) and their fusion proteins G6PDH-DI and DI-G6PDH were cloned from Geobacillus stearothermophilus, heterologouely expressed in E. coli and purified through its His-tag. The individual proteins G6PDH and DI have good thermostability and reactivity. However, the presence of DI in fusion proteins drastically decreased G6DPH activity. However, a mixture of G6PDH and a fusion protein G6PDH-DI not only restored G6PDH activity through the formation of heteromultimeric network but also facilitated substrate channeling between DI and G6PDH, especially at low enzyme concentrations. My researches would provide important building blocks for the on-going projects: high-yield hydrogen production through cell-free enzymatic pathways and electrical energy production through enzymatic fuel cells. / Master of Science

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento. / Perinatal hypoxia-ischemia (HI) is one of the major causes of mortality and chronic neurological diseases in newborns that can show permanent effects such as mental retardation, cerebral palsy, learning difficulty and epilepsy. It is probable that these impairs may be related to a delay in the neural development, astrogliosis and to the death of neurons and oligodendrocytes. Cognitive and functional deficits are related to degeneration of dopaminergic pathways and hippocampus. The enzyme tyrosine hydroxylase (TH) is a limiting step in the dopamine synthesis and its levels are impaired in HI insults. Nitric oxide (NO) is a diffusible gas that acts by modulating different systems and participates in several phenomena such as synaptic plasticity and neuromodulation in the central nervous system and is produced in higher levels in events of injury and inflamation as in the case of HI. This study aimed to evaluate the effects of HI on the motor behavior and to evaluate the development of brain structures related to this behavior as the substantia nigra (SN) and the hippocampal complex, using the model developed by Robinson and colleagues in 2005. HI was induced by clamping the uterine arteries of pregnant rats, for 45 minutes, on the eighteenth day of gestation (group HI). In a group of females, the surgery was performed, but no clamping of the arteries (group SHAM) was made. Assessment of motor behavior was performed with the ROTAROD test and open field test in animals of 45 days (P45) of age. The brains were processed histologically at ages P9, P16, P23 and P90, and then submitted to immunohistochemistry for TH and NADPH diaphorase (NADPH-d) histochemistry for evaluation of NOS. Our results demonstrated an apparent decrease in TH immunoreactivity in cell bodies in the SN at P16 in the HI group and an increase in immunoreactivity of the fibers in the SN pars reticulata at P23 with the presence of TH immunoreactive cell bodies at this same region in the HI group. We also showed an increase in the number of NADPH-d stained cells in the dentate gyrus in the HI group, at all ages, as also an increase in the intensity of staining in the Ammons horn (CA1 and CA3) at P9 in the HI group and, after that, a decrease in this staining at P23 and P90 in this same group. In the behavioral tests we observed a decrease in the motor activity in the HI group with a partial recovery all over the several sessions in the ROTAROD test, however this group did not reach the same performance as the SHAM group. HI animals did not show a higher level of anxiety when compared to SHAM animals, ruling out the hypothesis that anxiogenic factors may be impairing the results in the motor behavior tests. Our results showed that the model of uterine arteries clamping could be an important tool in the study of the effects of perinatal HI, by producing several consequences that are very similar to the effects observed in newborn children who suffered an HI event.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento. / Perinatal hypoxia-ischemia (HI) is one of the major causes of mortality and chronic neurological diseases in newborns that can show permanent effects such as mental retardation, cerebral palsy, learning difficulty and epilepsy. It is probable that these impairs may be related to a delay in the neural development, astrogliosis and to the death of neurons and oligodendrocytes. Cognitive and functional deficits are related to degeneration of dopaminergic pathways and hippocampus. The enzyme tyrosine hydroxylase (TH) is a limiting step in the dopamine synthesis and its levels are impaired in HI insults. Nitric oxide (NO) is a diffusible gas that acts by modulating different systems and participates in several phenomena such as synaptic plasticity and neuromodulation in the central nervous system and is produced in higher levels in events of injury and inflamation as in the case of HI. This study aimed to evaluate the effects of HI on the motor behavior and to evaluate the development of brain structures related to this behavior as the substantia nigra (SN) and the hippocampal complex, using the model developed by Robinson and colleagues in 2005. HI was induced by clamping the uterine arteries of pregnant rats, for 45 minutes, on the eighteenth day of gestation (group HI). In a group of females, the surgery was performed, but no clamping of the arteries (group SHAM) was made. Assessment of motor behavior was performed with the ROTAROD test and open field test in animals of 45 days (P45) of age. The brains were processed histologically at ages P9, P16, P23 and P90, and then submitted to immunohistochemistry for TH and NADPH diaphorase (NADPH-d) histochemistry for evaluation of NOS. Our results demonstrated an apparent decrease in TH immunoreactivity in cell bodies in the SN at P16 in the HI group and an increase in immunoreactivity of the fibers in the SN pars reticulata at P23 with the presence of TH immunoreactive cell bodies at this same region in the HI group. We also showed an increase in the number of NADPH-d stained cells in the dentate gyrus in the HI group, at all ages, as also an increase in the intensity of staining in the Ammons horn (CA1 and CA3) at P9 in the HI group and, after that, a decrease in this staining at P23 and P90 in this same group. In the behavioral tests we observed a decrease in the motor activity in the HI group with a partial recovery all over the several sessions in the ROTAROD test, however this group did not reach the same performance as the SHAM group. HI animals did not show a higher level of anxiety when compared to SHAM animals, ruling out the hypothesis that anxiogenic factors may be impairing the results in the motor behavior tests. Our results showed that the model of uterine arteries clamping could be an important tool in the study of the effects of perinatal HI, by producing several consequences that are very similar to the effects observed in newborn children who suffered an HI event.

Die NAD+-reduzierende Hydrogenase aus Ralstonia eutropha (SH) katalysiert die reversible H2-Oxidation in Verbindung mit der Reduktion von NAD+ in Gegenwart von Sauerstoff. Die bemerkenswerte O2-Toleranz des Enzyms wurde zuvor auf eine für [NiFe]-Hydrogenasen ungewöhnliche Struktur des Wasserstoff-spaltenden Zentrums zurückgeführt. Diese Hypothese wurde in dieser Arbeit mittels in situ-Spektroskopie an SH-haltigen Zellen widerlegt. Um die folgende Untersuchung der aus sechs Untereinheiten und mindestens acht Kofaktoren bestehenden SH zu erleichtern, wurde das Enzym mittels genetischer Methoden in seine beiden Module aufgeteilt. Das die H2-Oxidation katalysierende Hydrogenase-Modul beinhaltete ein FMN-Molekül, welches für die reduktive Reaktivierung des oxidativ modifizierten Zentrums benötigt wird. Das Diaphorase-Modul besaß ebenfalls ein FMN, und die Reduktion von NAD+ wurde von der Anwesenheit von O2 nicht beeinträchtigt. Neben Wasserstoff reagierte das [NiFe]-Zentrum der SH auch mit Sauerstoff. Dabei wurde sowohl Wasserstoffperoxid- als auch Wasser im Hydrogenase-Modul freigesetzt. Die Sauerstofftoleranz der SH basiert auf einer kontinuierlichen Reaktivierung des durch Sauerstoff oxidierten [NiFe]-Zentrums. Aufgrund der außergewöhnlichen Sauerstofftoleranz stellt die SH ein vielversprechendes System für die wasserstoffgetriebene Regeneration von NADH in gekoppelten enzymatischen Reaktionen dar. In dieser Arbeit wurde ein SH-Derivat durch rationale Mutagenese konstruiert, das in der Lage war, ebenso den Kofaktor NADP+ wasserstoffabhängig zu reduzieren. Durch Ganzzellansätze kann die zeitaufwändige und kostenintensive Proteinreinigung vermieden werden. Um die wasserstoffabhängige in-vivo-Kofaktorregeneration zu ermöglichen, wurde die SH in Pseudomonas putida heterolog produziert. Die in dieser Arbeit erzielten Ergebnisse sind sowohl für das molekulare Verständnis der H2-abhängigen Katalyse als auch für die biotechnologische Anwendung der O2-toleranten SH relevant. / The NAD+ reducing hydrogenase from Ralstonia eutropha (SH) catalyzes the reversible oxidation of hydrogen in connection with the reduction of NAD+ in the presence of oxygen. The remarkable oxygen tolerance was previously related to an unusual [NiFe] active site with four instead of two cyanide ligands. This hypothesis was rejected in this study by using in situ spectroscopy on SH containing cells. To simplify the investigation of the six-subunit and at least eight cofactors containing SH, the enzyme was separated into its two modules by genetic methods. The hydrogen oxidizing hydrogenase module contained one FMN molecule, which was required for the reductive reactivation of the oxidatively modified active site. The diaphorase module carried a second FMN. The reduction of NAD+ was not affected by the presence of oxygen. In addition to hydrogen, the [NiFe] center of the SH reacted with oxygen. Both hydrogen peroxide and water were released by the hydrogenase module. The oxygen tolerance of the SH is based on a continuous reactivation of the oxidized [NiFe] center. Due to the oxygen tolerance, the SH is a promising system for hydrogen based NADH regeneration in coupled enzymatic reactions. In this study a SH derivative was constructed by means of rational mutagenesis. The SH derivative was able to reduce the cofactor NADP+ by hydrogen oxidation. The time consuming and costly protein purification can be avoided by using whole cell approaches. In order to allow the hydrogen dependent in vivo cofactor regeneration, SH was heterologously produced in Pseudomonas putida. The results obtained in this study are relevant for the molecular understanding of hydrogen dependent catalysis and for the biotechnological application of the oxygen tolerant SH.

As rãs são utilizadas como modelos experimentais em diferentes situações experimentais. Uma delas é o estudo dos efeitos da seção do nervo isquiático (SNI) sobre o tecido nervoso. Essa ampla utilização desses animais como modelos experimentais justifica a realização de estudos que visam o conhecimento morfofuncional de seus tecidos. Inúmeros estudos mostram que, assim como nos mamíferos, o principal substrato energético no tecido nervoso de rãs é a glicose. Porém, é desconhecida a distribuição dos transportadores de glicose no tecido nervoso de rãs, bem como se a SNI altera esse transporte. Outra questão em aberto é se o lactato, cuja concentração está aumentada no plasma de rãs durante períodos de hibernação e após atividades motoras, é usado como substrato energético pelo tecido nervoso, o que está demonstrado em outras espécies de vertebrados. É desconhecida ainda no gânglio da raiz dorsal (GRD) de rãs a distribuição e os efeitos da SNT sobre a reação à nicotinamida adenina dinucleotídeo fosfato diaforase (NADPH-diaforase), enzima considerada equivalente a óxido nítrico sintase, responsável pela síntese de óxido nítrico, e a reação ao ácido periódico-reativo de Schiff (PAS), que indica a presença de mucopolissarídeos, incluindo o glicogênio, uma importante reserva energética no tecido nervoso de rãs. Desconhece-se também a distribuição e os efeitos da SNT sobre a imunorreatividade à serotonina, importante molécula com função neurotransmissora e/ou moduladora no sistema nervoso, tirosina hidroxilase, enzima limitante na síntese de catecolaminas, moléculas com diversos papéis fisiológicos, incluindo ação neurotransmissora e/ou neuromoduladora no tecido nervoso, e c-Fos, proteína considerada marcadora de ativação neural por estimulação nociva. Outras questões ainda em aberto são os efeitos da SNT sobre: a captação do análogo da glicose 1-14C 2-deoxi-D-glicose (14C-2-DG) e concentração plasmática de glicose e lactato; se os tipos II e III de células gliais satélites (CGSs), recentemente descritas no GRD de coelho, estão presentes nesse gânglio de rãs; e os efeitos da SNT sobre a ultraestrutura de CGSs e neurônios do GRD. Assim, o objetivo dessa tese foi determinar: 1) a ultraestrutura de neurônios e CGSs; 2) a distribuição das reações à NADPH-diaforase e PAS, e a imunoistoquímica à serotonina, tirosina hidroxilase, c-Fos e transportadores de glicose tipo 1 e 3; e 3) a captação de 14C-2-DG, na presença e ausência de lactato, em GRD de rãs Lithobates catesbianus com e sem SNI. A escolha pelos transportadores de glicose tipos 1 e 3 foi pelo fato de ocorrerem na membrana de endotélio, células gliais e de neurônios. Para a realização do estudo inicialmente 12 rãs Lithobates catesbianus, adultas, machos, com peso de 100-200g, que não sofreram qualquer manipulação cirúrgica foram mortas por decapitação e os gânglios das raízes dorsais (GRDs) do nervo isquiático retirados e preparados para análises ultraestrutural, histoquímica à NADPH-diaforase e PAS, e imunoistoquímica à serotonina, tirosina hidroxilase e transportadores de glicose dos tipos 1 e 3. Feito isso, 18 outras rãs, nas mesmas condições físicas, foram divididas em três grupos experimentais (n=6/grupo): controle (rãs que não sofreram qualquer manipulação cirúrgica), sham (rãs onde foram efetuados apenas os procedimentos para isolamento do nervo isquiático) e SNI (rãs que tiveram o nervo isquiático direito totalmente seccionado em seu tronco comum). Esses animais foram mortos três dias após a intervenção cirúrgica e seus GRDs do nervo isquiático usados para demonstrar os efeitos da secção nervosa sobre a ultraestrutura, a reação à NADPH-diaforase, e a imunoistoquímica à serotonina, tirosina hidroxilase, c-Fos e transportadores de glicose dos tipos 1 e 3 no GRD. Outros 20 animais, divididos nos mesmos grupos experimentais, foram usados para demonstrar os efeitos da SNI sobre a captação de 14C-2-DG, na presença ou ausência de lactato, e a taxa de produção de 14CO2 a partir de 14C-L-lactato e de 14C-glicose no GRD. Essas rãs foram usadas ainda para demonstrar os efeitos da denervação periférica sobre a concentração plasmática de glicose e lactato. Nossos resultados mostraram que os neurônios sensoriais do GRD de rã Lithobates catesbianus tiveram distribuição, diâmetro e morfologia que foi similar àquela descrita para essas células em gânglio de mamíferos. As CGSs apresentaram morfologia similar àquela descrita para essas células em gânglios de outras espécies de vertebrados. As células dos tipos II e III, observadas no GRD de coelho, não ocorreram no GRD de Lithobates catesbianus. O padrão de atividade à NADPH-diaforase e a distribuição da imunorreatividade à serotonina, tirosina hidroxilase e Glut 1 e 3 foram também similares ao descrito em mamíferos. Pela primeira vez foi demonstrada, em anfíbios, a presença de reação à NADPH-diaforase em CGCs do GRD. A captação de 14C-2-DG foi reduzida quando o lactato foi acrescentado ao meio de incubação. As alterações induzidas pela SNI foram também similares àquelas descritas nos mamíferos. Houve acréscimo no número de mitocôndrias, retículo endoplasmático, ribossomas e filamentos no citoplasma das CGSs, mais neurônios e CGCs com reação positiva à NADPH-diaforase, um maior número de prolongamentos imunorreativos à tirosina hidroxilase em torno de somas de neurônios sensoriais, e mais núcleos neuronais imunorreativos a c-Fos. Nenhuma alteração ocorreu na imunorreatividade a serotonina e transportadores de glicose. Houve aumento na captação de 14C-2-DG, que foi reduzido quando o lactato foi acrescentado ao meio de incubação. Porém, a formação de 14CO2 a partir de 14C-L-lactato e de 14C-glicose não alterou nessas condições. Todavia, diferentemente dos mamíferos, a SNI não provocou mudança no número de CGCs no GRD, mostrando uma peculiaridade na resposta das rãs à SNI. Assim, nosso estudo reforça o uso de rãs como modelo experimental para estudo dos efeitos da SNI, um modelo de dor fantasma, sobre o tecido nervoso. Porém, dada a diferença peculiar ocorrida no GRD de rãs com SNI, é evidente a necessidade de mais conhecimento dos efeitos dessa situação experimental nesses animais. / Frogs have been used as experimental models in different experimental situations. One of these is the study of the effects of the sciatic nerve transection (SNT) on the nerve tissue. The wide use of these animals as experimental models justifies the studies aimed at morphofunctionally understanding of their tissues. Numerous studies have shown that glucose is the main energy substrate in the nerve tissue of frogs as well as in mammals. However, the distribution of glucose transporters in the nerve tissue of frogs is unknown as well as whether SNT alters such transportation. Another unanswered question is whether the lactate, whose concentration is increased in the frog plasma during hibernation periods and after motor activities, is used as an energy substrate by the nerve tissue, which has been demonstrated in other vertebrate species. In the dorsal root ganglion (DRG) cells of frogs are still unknown the distribution and effects of SNT on the reaction of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase), an enzyme that is considered equivalent to nitric oxide synthase, responsible for the synthesis of nitric oxide, and on the reaction of periodic acid-Schiff (PAS), which indicates the presence of mucopolysaccharides, including glycogen, an important energy reserve in frog nerve tissue. Moreover, the distribution and effects of SNT on immunoreactivity to serotonin, an important molecule that functions as a neurotransmitter and / or neuromodulator in the nervous system, tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, molecules with various physiological roles, including neurotransmitter and / or neuromodulator action in the nerve tissue, and c-Fos, a protein that is regarded as a marker of neuronal activation by noxious stimulation are also unknown. Other questions regarding is the effect of SNT on the uptake of glucose analogue 2-Deoxy-D-glucose-1-14C (14C-2-DG) and glucose and lactate concentration plasma; whether the types II and III of satellite glial cells (SGCs), recently described in rabbit DRG, are present in this ganglion of frogs; and the effects of SNT on the ultrastructure of SGCs and DRG neurons remain unanswered as well. Thus, this thesis aimed to determine: 1) the ultrastructure of neurons and SGCs; 2) the distribution of NADPH-diaphorase and PAS reaction, and immunohistochemistry for serotonin, tyrosine hydroxylase, c-Fos and glucose transporters types 1 and 3; and 3) the uptake of 2-DG-14C, in the presence and absence of lactate, in DRG of frogs, Lithobates catesbianus, with and without SNT. Glucose transporters types 1 and 3 were chosen because they occur in the membrane of endothelial cells, glial cells and neurons. Initially, 12 adult male frogs, Lithobates catesbianus, weighing 100-200g, not having undergone any previous surgical manipulation, were killed by decapitation. The DRGs of the sciatic nerve were removed and prepared for ultrastructural analysis, histochemistry of NADPH-diaphorase and PAS, and immunohistochemistry for serotonin, tyrosine hydroxylase and glucose transporters types 1 and 3. After that, 18 other frogs in the same physical conditions were divided into three experimental groups (n = 6/group): control group (frogs not subjected to any surgical manipulation), sham (frogs in which only surgical procedures for isolating the sciatic nerve were performed), and SNT (frogs in which the right sciatic nerve was completely transected). These animals were killed three days after the procedure, and their sciatic nerve DRGs used to demonstrate the effects of nerve transection on the ultrastructure, NADPH-diaphorase reaction, and immunohistochemical serotonin, tyrosine hydroxylase, c-Fos and glucose transporters types 1 and 3 in the DRG. Other 20 animals, divided into the same experimental groups, were used to demonstrate the effects of SNI on the uptake of 14C-2-DG in the presence or absence of lactate, the production rate of 14CO2 from 14C-L-lactate and 14C-glucose in the DRG. These frogs were used to further demonstrate the effects of peripheral denervation on plasma glucose and lactate levels. Our results have demonstrated that sensory neurons of bullfrog, Lithobates catesbianus, DRG showed distribution, diameter and morphology similar to those described for these ganglion cells in mammals. The CGSs showed morphology similar to that described for these cells in the lymph nodes of other vertebrate species. Cells types II and III, observed in rabbit DRG did not occur in the Lithobates catesbianus DRG. The pattern of NADPH-diaphorase activity and distribution of immunoreactivity of serotonin, tyrosine hydroxylase and Glut 1 and 3 were also similar to those described in mammals. For the first time, it has been demonstrated the presence of NADPH-diaphorase reaction on SGCs of DRG in amphibians. The uptake of 14C-2-DG was reduced when lactate was added to the incubation medium. SNT-induced changes were also similar to those ones described in mammals. There was an increase in the number of mitochondria, endoplasmic reticulum, ribosomes and filaments in the SGCs cytoplasm; more neurons and SGCs with positive reaction to NADPH-diaphorase; a greater number of tyrosine hydroxylase immunoreactive extensions around body sensory neurons; and more c-Fos immunoreactivity in neuronal nuclei. No changes occurred in serotonin immunoreactivity and glucose transporters. There was an increase in the uptake of 14C-2-DG, which was reduced when lactate was added to the incubation medium. However, the formation of 14C-2-DG from 14C-L-lactato and glucose did not change under these conditions. Unlike mammals, SNT caused no change in the number of SGCs in DRG, showing a peculiarity in the response of frogs to SNT. Therefore, our study supports the use of frogs as an experimental model to study the effects of SNT, a model of phantom pain on the nerve tissue. However, given the peculiar differences occurred in the DRG of frogs with SNT, it is clearly necessary to carry out further studies to better understand the effects of an experimental situation like this in such animals.

As rãs são utilizadas como modelos experimentais em diferentes situações experimentais. Uma delas é o estudo dos efeitos da seção do nervo isquiático (SNI) sobre o tecido nervoso. Essa ampla utilização desses animais como modelos experimentais justifica a realização de estudos que visam o conhecimento morfofuncional de seus tecidos. Inúmeros estudos mostram que, assim como nos mamíferos, o principal substrato energético no tecido nervoso de rãs é a glicose. Porém, é desconhecida a distribuição dos transportadores de glicose no tecido nervoso de rãs, bem como se a SNI altera esse transporte. Outra questão em aberto é se o lactato, cuja concentração está aumentada no plasma de rãs durante períodos de hibernação e após atividades motoras, é usado como substrato energético pelo tecido nervoso, o que está demonstrado em outras espécies de vertebrados. É desconhecida ainda no gânglio da raiz dorsal (GRD) de rãs a distribuição e os efeitos da SNT sobre a reação à nicotinamida adenina dinucleotídeo fosfato diaforase (NADPH-diaforase), enzima considerada equivalente a óxido nítrico sintase, responsável pela síntese de óxido nítrico, e a reação ao ácido periódico-reativo de Schiff (PAS), que indica a presença de mucopolissarídeos, incluindo o glicogênio, uma importante reserva energética no tecido nervoso de rãs. Desconhece-se também a distribuição e os efeitos da SNT sobre a imunorreatividade à serotonina, importante molécula com função neurotransmissora e/ou moduladora no sistema nervoso, tirosina hidroxilase, enzima limitante na síntese de catecolaminas, moléculas com diversos papéis fisiológicos, incluindo ação neurotransmissora e/ou neuromoduladora no tecido nervoso, e c-Fos, proteína considerada marcadora de ativação neural por estimulação nociva. Outras questões ainda em aberto são os efeitos da SNT sobre: a captação do análogo da glicose 1-14C 2-deoxi-D-glicose (14C-2-DG) e concentração plasmática de glicose e lactato; se os tipos II e III de células gliais satélites (CGSs), recentemente descritas no GRD de coelho, estão presentes nesse gânglio de rãs; e os efeitos da SNT sobre a ultraestrutura de CGSs e neurônios do GRD. Assim, o objetivo dessa tese foi determinar: 1) a ultraestrutura de neurônios e CGSs; 2) a distribuição das reações à NADPH-diaforase e PAS, e a imunoistoquímica à serotonina, tirosina hidroxilase, c-Fos e transportadores de glicose tipo 1 e 3; e 3) a captação de 14C-2-DG, na presença e ausência de lactato, em GRD de rãs Lithobates catesbianus com e sem SNI. A escolha pelos transportadores de glicose tipos 1 e 3 foi pelo fato de ocorrerem na membrana de endotélio, células gliais e de neurônios. Para a realização do estudo inicialmente 12 rãs Lithobates catesbianus, adultas, machos, com peso de 100-200g, que não sofreram qualquer manipulação cirúrgica foram mortas por decapitação e os gânglios das raízes dorsais (GRDs) do nervo isquiático retirados e preparados para análises ultraestrutural, histoquímica à NADPH-diaforase e PAS, e imunoistoquímica à serotonina, tirosina hidroxilase e transportadores de glicose dos tipos 1 e 3. Feito isso, 18 outras rãs, nas mesmas condições físicas, foram divididas em três grupos experimentais (n=6/grupo): controle (rãs que não sofreram qualquer manipulação cirúrgica), sham (rãs onde foram efetuados apenas os procedimentos para isolamento do nervo isquiático) e SNI (rãs que tiveram o nervo isquiático direito totalmente seccionado em seu tronco comum). Esses animais foram mortos três dias após a intervenção cirúrgica e seus GRDs do nervo isquiático usados para demonstrar os efeitos da secção nervosa sobre a ultraestrutura, a reação à NADPH-diaforase, e a imunoistoquímica à serotonina, tirosina hidroxilase, c-Fos e transportadores de glicose dos tipos 1 e 3 no GRD. Outros 20 animais, divididos nos mesmos grupos experimentais, foram usados para demonstrar os efeitos da SNI sobre a captação de 14C-2-DG, na presença ou ausência de lactato, e a taxa de produção de 14CO2 a partir de 14C-L-lactato e de 14C-glicose no GRD. Essas rãs foram usadas ainda para demonstrar os efeitos da denervação periférica sobre a concentração plasmática de glicose e lactato. Nossos resultados mostraram que os neurônios sensoriais do GRD de rã Lithobates catesbianus tiveram distribuição, diâmetro e morfologia que foi similar àquela descrita para essas células em gânglio de mamíferos. As CGSs apresentaram morfologia similar àquela descrita para essas células em gânglios de outras espécies de vertebrados. As células dos tipos II e III, observadas no GRD de coelho, não ocorreram no GRD de Lithobates catesbianus. O padrão de atividade à NADPH-diaforase e a distribuição da imunorreatividade à serotonina, tirosina hidroxilase e Glut 1 e 3 foram também similares ao descrito em mamíferos. Pela primeira vez foi demonstrada, em anfíbios, a presença de reação à NADPH-diaforase em CGCs do GRD. A captação de 14C-2-DG foi reduzida quando o lactato foi acrescentado ao meio de incubação. As alterações induzidas pela SNI foram também similares àquelas descritas nos mamíferos. Houve acréscimo no número de mitocôndrias, retículo endoplasmático, ribossomas e filamentos no citoplasma das CGSs, mais neurônios e CGCs com reação positiva à NADPH-diaforase, um maior número de prolongamentos imunorreativos à tirosina hidroxilase em torno de somas de neurônios sensoriais, e mais núcleos neuronais imunorreativos a c-Fos. Nenhuma alteração ocorreu na imunorreatividade a serotonina e transportadores de glicose. Houve aumento na captação de 14C-2-DG, que foi reduzido quando o lactato foi acrescentado ao meio de incubação. Porém, a formação de 14CO2 a partir de 14C-L-lactato e de 14C-glicose não alterou nessas condições. Todavia, diferentemente dos mamíferos, a SNI não provocou mudança no número de CGCs no GRD, mostrando uma peculiaridade na resposta das rãs à SNI. Assim, nosso estudo reforça o uso de rãs como modelo experimental para estudo dos efeitos da SNI, um modelo de dor fantasma, sobre o tecido nervoso. Porém, dada a diferença peculiar ocorrida no GRD de rãs com SNI, é evidente a necessidade de mais conhecimento dos efeitos dessa situação experimental nesses animais. / Frogs have been used as experimental models in different experimental situations. One of these is the study of the effects of the sciatic nerve transection (SNT) on the nerve tissue. The wide use of these animals as experimental models justifies the studies aimed at morphofunctionally understanding of their tissues. Numerous studies have shown that glucose is the main energy substrate in the nerve tissue of frogs as well as in mammals. However, the distribution of glucose transporters in the nerve tissue of frogs is unknown as well as whether SNT alters such transportation. Another unanswered question is whether the lactate, whose concentration is increased in the frog plasma during hibernation periods and after motor activities, is used as an energy substrate by the nerve tissue, which has been demonstrated in other vertebrate species. In the dorsal root ganglion (DRG) cells of frogs are still unknown the distribution and effects of SNT on the reaction of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase), an enzyme that is considered equivalent to nitric oxide synthase, responsible for the synthesis of nitric oxide, and on the reaction of periodic acid-Schiff (PAS), which indicates the presence of mucopolysaccharides, including glycogen, an important energy reserve in frog nerve tissue. Moreover, the distribution and effects of SNT on immunoreactivity to serotonin, an important molecule that functions as a neurotransmitter and / or neuromodulator in the nervous system, tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, molecules with various physiological roles, including neurotransmitter and / or neuromodulator action in the nerve tissue, and c-Fos, a protein that is regarded as a marker of neuronal activation by noxious stimulation are also unknown. Other questions regarding is the effect of SNT on the uptake of glucose analogue 2-Deoxy-D-glucose-1-14C (14C-2-DG) and glucose and lactate concentration plasma; whether the types II and III of satellite glial cells (SGCs), recently described in rabbit DRG, are present in this ganglion of frogs; and the effects of SNT on the ultrastructure of SGCs and DRG neurons remain unanswered as well. Thus, this thesis aimed to determine: 1) the ultrastructure of neurons and SGCs; 2) the distribution of NADPH-diaphorase and PAS reaction, and immunohistochemistry for serotonin, tyrosine hydroxylase, c-Fos and glucose transporters types 1 and 3; and 3) the uptake of 2-DG-14C, in the presence and absence of lactate, in DRG of frogs, Lithobates catesbianus, with and without SNT. Glucose transporters types 1 and 3 were chosen because they occur in the membrane of endothelial cells, glial cells and neurons. Initially, 12 adult male frogs, Lithobates catesbianus, weighing 100-200g, not having undergone any previous surgical manipulation, were killed by decapitation. The DRGs of the sciatic nerve were removed and prepared for ultrastructural analysis, histochemistry of NADPH-diaphorase and PAS, and immunohistochemistry for serotonin, tyrosine hydroxylase and glucose transporters types 1 and 3. After that, 18 other frogs in the same physical conditions were divided into three experimental groups (n = 6/group): control group (frogs not subjected to any surgical manipulation), sham (frogs in which only surgical procedures for isolating the sciatic nerve were performed), and SNT (frogs in which the right sciatic nerve was completely transected). These animals were killed three days after the procedure, and their sciatic nerve DRGs used to demonstrate the effects of nerve transection on the ultrastructure, NADPH-diaphorase reaction, and immunohistochemical serotonin, tyrosine hydroxylase, c-Fos and glucose transporters types 1 and 3 in the DRG. Other 20 animals, divided into the same experimental groups, were used to demonstrate the effects of SNI on the uptake of 14C-2-DG in the presence or absence of lactate, the production rate of 14CO2 from 14C-L-lactate and 14C-glucose in the DRG. These frogs were used to further demonstrate the effects of peripheral denervation on plasma glucose and lactate levels. Our results have demonstrated that sensory neurons of bullfrog, Lithobates catesbianus, DRG showed distribution, diameter and morphology similar to those described for these ganglion cells in mammals. The CGSs showed morphology similar to that described for these cells in the lymph nodes of other vertebrate species. Cells types II and III, observed in rabbit DRG did not occur in the Lithobates catesbianus DRG. The pattern of NADPH-diaphorase activity and distribution of immunoreactivity of serotonin, tyrosine hydroxylase and Glut 1 and 3 were also similar to those described in mammals. For the first time, it has been demonstrated the presence of NADPH-diaphorase reaction on SGCs of DRG in amphibians. The uptake of 14C-2-DG was reduced when lactate was added to the incubation medium. SNT-induced changes were also similar to those ones described in mammals. There was an increase in the number of mitochondria, endoplasmic reticulum, ribosomes and filaments in the SGCs cytoplasm; more neurons and SGCs with positive reaction to NADPH-diaphorase; a greater number of tyrosine hydroxylase immunoreactive extensions around body sensory neurons; and more c-Fos immunoreactivity in neuronal nuclei. No changes occurred in serotonin immunoreactivity and glucose transporters. There was an increase in the uptake of 14C-2-DG, which was reduced when lactate was added to the incubation medium. However, the formation of 14C-2-DG from 14C-L-lactato and glucose did not change under these conditions. Unlike mammals, SNT caused no change in the number of SGCs in DRG, showing a peculiarity in the response of frogs to SNT. Therefore, our study supports the use of frogs as an experimental model to study the effects of SNT, a model of phantom pain on the nerve tissue. However, given the peculiar differences occurred in the DRG of frogs with SNT, it is clearly necessary to carry out further studies to better understand the effects of an experimental situation like this in such animals.