Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [105]-131).

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "May 2002." Typescript. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 87-95).

Ca2+ is an important second messenger which modulates a variety of signalling pathways in both excitable and non-excitable cells. In the CNS, Ca2+ plays an important role in neurons both physiologically and pathologically. Ca2+ influx following synaptic activity, is important in development, plasticity, redox balance, as well as in neuroprotection, largely through activation of pro-survival pathways downstream of synaptic NMDAR activation, including upregulation of antioxidant defences. However, excessive Ca2+ influx in neurons leads to neuronal damage and excitotoxicity, in which mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (Mcu) resulting in mitochondrial dysfunction is a key player. Excitotoxicity occurs due to glutamate efflux from astrocytes following stroke, traumatic brain injury and in chronic neurodegenerative diseases, leading to excessive neuronal NMDAR activation and triggering of its downstream pro-death pathways. This thesis focuses on understanding the pro-survival and pro-death effects of signalling pathways activated by Ca2+ in neurons, as well as the potential effect of neuronal synaptic activity on influencing neuroprotective gene transcription in astrocytes. I investigated the role of AMPK, a master regulator of metabolism, in NMDA excitotoxicity in cortical neurons as a potential downstream effector of Mcu-dependent excitotoxic death; and found the deletion of AMPKα1/2 to be neuroprotective against NMDA-mediated excitotoxicity, however I found AMPK activation to be independent of Mcu. I also investigated the expression pattern of Mcu and other mitochondrial calcium regulatory genes (MCRGs), and found MCRGs to be differentially expressed in different neural cells (primary neurons vs astrocytes), and neuronal subtypes (CA1 vs CA3 region of the hippocampus), suggesting differing dependence on the various MCRGs in mitochondrial Ca2+ handling in these cell types. A better functional understanding of these genes will allow for investigation of their importance in mitochondrial Ca2+ handling, including their potential role in excitotoxicity. I next investigated the neuroprotective effects of synaptic activity induced Ca2+ influx, focusing on antioxidant target genes of Nrf2, a transcription factor and major regulator of antioxidant genes. I found that unlike astrocytes, neurons express very low levels of Nrf2. However, synaptic activity increased the expression of several Nrf2 target genes in neurons, independently of astrocytes and Nrf2. Additionally, I found no effect of synaptic activity on increasing Nrf2 protein levels, despite previous reports in literature of Nrf2 pathway activation following synaptic activity. Finally, using RNA-seq I identified a list of genes strongly upregulated by a known Nrf2 activator in astrocytes, and found no evidence that neuronal activity triggers expression of these genes independently of neurons, providing further evidence that neuronal activity does not activate the Nrf2 pathway in astrocytes. This suggests that synaptic activity via pathways activated by Ca2+ signalling provides neurons with cell-autonomous antioxidant defences, independently of Nrf2; thus providing a distinct pathway for antioxidant defences in neurons from the Nrf2 pathway, which is activated in astrocytes providing neurons with non-cell autonomous antioxidant support. These results give us further insight into the mechanisms that underlie synaptic and non-synaptic Ca2+ signalling pathways mediating neuronal survival and death, which could help in identifying therapeutic targets to combat excitotoxicity and oxidative stress in various neurological diseases.

Orientador: Alexandre Leite Rodrigues de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T22:49:48Z (GMT). No. of bitstreams: 1 Marques_KarinadeBrito_D.pdf: 8050913 bytes, checksum: f9c7d621391d6f3f99413a9c27749530 (MD5) Previous issue date: 2007 / Resumo: Durante o curso da encefalomielite autoimmune experimental ocorre uma grave redução das funções motoras e sensitivas. Esses eventos têm sido classicamente atribuídos ao processo desmielinizante da doença. Em ratos, os sinais clínicos da doença desaparecem 5 dias após completa tetraplegia, indicando que o processo desmielinizante não é a única causa da rápida evolução da doença. Assim sendo, investigamos as alterações sinaptológicas e o processo inflamatório induzidos pela encefalomielite autoimune experimental (EAE) em motoneurônios medulares e sua relação com o surto e remissão da doença. Para esse estudo, foram utilizados ratos Lewis, fêmeas de 7 semanas. Os animais foram induzidos à EAE por meio de dose única de proteína básica de mielina emulsificada com adjuvante completo de Freund e sacrificados no 13º dia após indução (surto grau 3) e no 26º dia (remissão da doença). Também, para investigar a possibilidade de que o tratamento com acetato de glatirâmer, uma droga imunomoduladora baseada na estrutura de aminoácidos da proteína básica de mielina, interfira no processo de plasticidade sináptica, os animais foram induzidos à EAE, tratados com AG diariamente e sacrificados após 2 semanas. Os grupos experimentais foram divididos em: estudo da aposição sináptica durante surto e remissão da doença e tratamento dos animais induzidos à EAE com AG. Assim, os espécimes foram processados para análise através de imunohistoquímica e microscopia eletrônica de transmissão. Nossos resultados indicaram que os componentes gliais (astrócitos e microglia), estimulados pela inflamação, desempenham papel ativo no processo de retração sináptica em motoneurônios alfa. Apresentamos evidências de que a eliminação de terminais sinápticos contribui para a perda da função motora observada no curso da doença e que o imunomodulador AG não só possui efeito antiinflamatório, mas também influencia diretamente na plasticidade de elementos neurais no microambiente medular. Reforçam, também, que um processo agudo de inflamação pode colaborar diretamente para a recuperação e sobrevivência neuronal, uma vez que as células inflamatórias produzem citocinas e fatores neurotróficos no microambiente medular / Abstract: During the course of experimental autoimmune encephalomyelitis, a massive loss of motor and sensitive function occurs, which has been classically attributed to the demyelination process. In rats, the clinical signs disappear within 5 days following complete tetraplegia, indicating that demyelination might not be the only cause for the rapid evolution of the disease. The immunomodulador glatiramer acetate (GA) has been shown significantly reduce the seriousness of the symptoms during the exacerbation of the disease. However, little is known about its effects on the spinal motoneurons and on their afferents. The present work investigated the occurrence of experimental autoimmune encephalomyelitis-induced changes of the synaptic covering of spinal motoneurons during exacerbation and after remission and investigated whether GA has a direct influence on synapse plasticity and on the deafferentiation of motoneurons during the course of EAE in rats. Lewis rats were subjected to EAE associated with GA or placebo treatment. The animals were sacrificed after fifteen days of treatment. For the both cases the spinal cords was processed for immunohistochemical analysis (IH) and electron transmission microscopy. The terminals were typed with transmission electron microscopy as C-, F- and Stype. Immunohistochemical analysis of synaptophysin, glial fibrillary acidic protein and the microglia/macrophage marker F4/80 were also used in order to draw a correlation between the synaptic changes and the glial reaction. The ultrastructural analysis showed that, during exacerbation, there was a strong retraction of both F- and S-type terminals. In this sense, both the covering as well as the length of the remaining terminals suffered great reductions. However, the retracted terminals rapidly returned to apposition, although the mean length remained shorter. A certain level of sprouting may have occurred as, after remission, the number of F-terminals was greater than in the control group. The immunohistochemical analysis showed that the peak of synaptic loss was coincident with an increased macro- and microglial reaction. Interestingly, although the GA treatment preserved synaptophysin labelling, it did not significantly reduce the glial reaction, indicating that inflammatory activity was still present. Our results suggest that the major changes occurring in the spinal cord network during the time course of the disease may contribute significantly to the origin of the clinical signs as well as help to explain their rapid recovery and that the immunomodulator GA has a direct influence on the stability of nerve terminals in the spinal cord, which in turn may contribute to its neuroprotective effects during the course of multiple sclerosis / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Orientador: Maria Alice da Cruz-Hofling / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T19:23:15Z (GMT). No. of bitstreams: 1 Zago_GabrielaMariotoni_M.pdf: 5451987 bytes, checksum: 84e15c6e492816ce6e4cc4c92760e544 (MD5) Previous issue date: 2007 / Resumo: A barreira hematoencefálica (BHE) é a principal estrutura controladora da manutenção da homeostase do SNC. A perda da integridade da BHE em respostas inflamatórias do SNC, desencadeada por agentes neurotóxicos, têm sido associadas ao desenvolvimento de sinais neurológicos. O veneno da aranha Phoneutria nigriventer (PNV) produz sinais e sintomas excitatórios em humanos e sua ação neurotóxica sugere habilidade potencial em alterar a permeabilidade da BHE. Nesse trabalho, o PNV foi utilizado como ferramenta para avaliar a susceptibilidade da BHE em diferentes regiões anatômicas cerebrais de ratos. Após injeção sistêmica do PNV (0.85 mg/Kg in 0.5 ml), os ratos anestesiados foram perfundidos 1, 2 e 5 h após a injeção, com solução fixadora à qual foi adicionado traçador extracelular eletron-opaco. Córtex motor fronto-parietal, substância cinzenta periaquedutal, núcleos da base e amigdala foram dissecados e processados para microscopia eletrônica de transmissão. O estado funcional da BHE foi avaliado considerando evidências do vedamento da barreira (edema vasogênico e extravasamento do traçador) e a resposta de elementos do tecido circunjacente (astrócitos, terminais sinápticos, populações de células). Além disso, foi investigada a expressão das proteínas GFAP, o principal filamento intermediário dos astrócitos, proteína S100, uma família de proteínas ligantes de cálcio e as citocinas pró-inflamatórias, IFN-? e TNF-a, através de marcação imunohistoquímica, no hipocampo e cerebelo. Logo após a administração do PNV, os animais mostraram sinais indicativos de envolvimento do SNC, SNP e SNA. Nossos resultados mostraram que todas as regiões analisadas apresentaram sinais morfológicos de reação defensiva, tais como migração de micróglia perivascular reativa, pés-vasculares astrocitário edemaciados e macrófagos ativos circulantes. Entretanto, apenas o córtex motor fronto-parietal mostrou número significante de vasos afetados em relação aos controles e às outras áreas anatômicas (1 h p.i.). Nos grupos controle, uma expressão basal de GFAP e S100B foi mantida inalterada durante os períodos de observação, enquanto nenhuma produção fisiológica das proteínas TNFa e INF? ocorreu. Por outro lado, a análise dos grupos tratados com PNV mostrou que, variavelmente, todas as proteínas investigadas aumentaram sua expressão no cerebelo e hipocampo ao longo do tempo após a injeção do veneno. O aumento da GFAP no cerebelo foi mais precoce e mais forte do que no hipocampo. Essa gliose mais evidente no cerebelo, provavelmente justifica estudos prévios, onde o extravasamento do traçador foi menor nessa região do que hipocampo, demonstrando assim, uma maior resistência da BHE do cerebelo. Outros mecanismos moleculares envolvidos poderiam ser a expressão de citocinas pró-inflamatórias TNFa e INF?, cuja modulação diferente em hipocampo e cerebelo de animais tratados com PNV, poderia também ter papel nas diferenças de permeabilidade da BHE vistas em ambas as áreas após PNV. Nosso trabalho dá suporte à hipótese de que os sinais e sintomas apresentados pelos animais durante o intervalo de tempo, após a injeção de PNV e o sacrifício, refletem alterações fisiológicas em curso, que por sua vez se revela ao nível histológico e ultraestrutural no desigual envolvimento da BHE nas diferente regiões cerebrais analisadas. Os vasos do córtex motor fronto parietal foram mais afetados pelo PNV, do que as demais regiões, confirmando a existência de diferenças regionais na capacidade do tecido local de mediar eventos requeridos para que ocorram as alterações da permeabilidade da BHE e para a invasão de populações celulares. O veneno de Phoneutria nigriventer representa uma importante substância natural, cuja complexa composição pode ser explorada em relação à ação de drogas que agem no SNC / Abstract: The blood¿brain barrier (BBB) is of pivotal importance to maintain homeostasis of the CNS, as it closely regulates the composition of the interstitial fluid in the brain. The loss of BBB integrity in CNS inflammatory responses triggered by neurotoxic agents has been associated with the development of neurological signs. Phoneutria nigriventer armed spider venom (PNV) produces excitatory signals and symptoms in humans, and its recognized neurotoxic action suggests a potential ability to alter BBB permeability. In this work, the PNV was used as tool to analyzing the BBB susceptibility of different rat brain anatomic regions. After PNV systemic injection (0.85 mg/ Kg in 0.5 ml) the rats were perfused at 1, 2 and 5 h post-injection (p.i.) with fixative solution to which had been added an electro-opaque extracellular tracer. Frontal-Parietal Motor Cortex, Periaqueductal Gray Matter, Base Nucleus and Amygdala were dissected and processed for routine transmission electron microscopy. The functional state of the BBB was evaluated considering evidences of the tightness of the barrier (vasogenic edema and tracer extravasation) and the response of elements of circumjacent tissue (astrocytes, synaptic endings, cells population). Besides, it was investigated the expression of the GFAP, the major intermediate filament of astrocytes, S100 protein, a family of calcium-binding proteins, and IFN-? and TNF-a pro-inflammatory cytokines, through imunohistochemistry labeling, in the hippocampus and cerebellum. Soon after PNV dministration the animals showed clinical signs indicative of peripheral, autonomic and central nervous system involvement. Our findings showed that all regions analyzed presented morphological signs of defensive reaction, such as migrating reactive perivascular microglia, swollen astrocytes end-feet and circulating active macrophages. However, only FPMC showed significant number of affected vessels in relation to controls and the other anatomic areas (1 h p.i.). A basal expression of GFAP and S100 was maintained unaltered along the periods of observation, whereas none physiologic production of TNFa and INF? proteins has occurred in control groups. In contrast, analysis of the PNV-treated groups showed that all investigated proteins variably enhanced its expression along the time-course after venom injection in cerebellum and hippocampus. The increase of GFAP in cerebellum is more precocious and stronger than in hippocampus. A more prominent reactive gliosis by cerebellum over hippocampus would be supposedly one of the molecular events underlying the previous findings showing to be cerebellum BBB more resistant to leakage than hippocampus. Other possible molecular mechanism involved would be the expression of proinflammatory TNFa and INF? cytokines, whose different modulation in cerebellum and hippocampus of PNV-treated animals could be also involved in differences of permeation of BBB seen in both areas. Our study further support the idea that the symptomatic interval after systemic P. nigriventer spider venom injection is characterized by sequential physiologic changes that are reflected in histological and ultrastructural preparations and reveal that BBB impairment is unequal in different anatomical brain areas. The Fronto-Parietal Motor Cortex vessels are more targeted for PNV, confirming the existence of regional differences in the capacity of the local tissue of mediating the events required for the changes of BBB permeability and for cell invasion. Phoneutria nigriventer venom represents an important natural substance, whose complex composition should be explored in terms of CNS acting drugs / Mestrado / Farmacologia / Mestre em Farmacologia

No description available.

Despite its numerous side effects, morphine and the opioids derived from this drug, belong among the only effective options for treatment of pain linked to oncological illness. The effect of opioids on the efficiency of cytostatics in vitro has been the subject of many papers but the results are often contradictory, which could be probably caused by the great variability of experimental models and approaches. Some recent studies indicate that the consequences of activation of opioid signaling in astrocytes display certain differences from other cell types. Glioblastoma multiforme, the tumor derived from astrocytes, belong among those with the worst prognosis, mostly for the frequent resistance to cytostatics. In this thesis we focused on the effect of morphine, methadone and DADLE on the efficiency of cytostatics of temozolomide, doxorubicin and vincristine on the cell lines C6 and CCF-STTG1 derived from glioblastomas. Also, we examined the effect of the above mentioned opioids on the level of oxidative cellular stress and using N-acetylcysteine, a ROS scavenger, we verified the role of oxidative stress in cellular systems activated by the effect of the mentioned opioids on the efficiency of cytostatics. We also assessed opioid receptors and the receptor TLR4 in the examined cell lines. The...

Our previous results showed that oligodendrocyte development is regulated by both nociceptin and its G-protein coupled receptor, the nociceptin/orphanin FQ receptor (NOPR). The present in vitro and in vivo findings show that nociceptin plays a crucial conserved role in both human and rodent brain astrocytes, regulating the levels of the glutamate/aspartate transporter GLAST/EAAT1. This nociceptin-mediated response takes place during a critical developmental window that coincides with astrocyte maturation and synapse formation. GLAST/EAAT1 upregulation by nociceptin is mediated by NOPR and the downstream participation of a complex signaling cascade that involves the interaction of several kinase systems, including PI-3K/AKT, mTOR and JAK. Because GLAST is the main glutamate transporter during brain maturation, these novel findings suggest that nociceptin plays a crucial role in regulating the function of early astrocytes and their capacity to support glutamate homeostasis in the developing brain.

No description available.