Wan Leong Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 80-83). / Acknowledgments --- p.i / Bibliography --- p.ii / Contents --- p.iii / Abstract --- p.iv / Abbreviations --- p.v / Chapter I --- Introduction / Chapter I-1 --- General Background of Pseudo-sugar --- p.1 / Chapter I-2 --- Monocarba-sugar --- p.2 / Chapter I-3 --- Dicarba-sugar --- p.4 / Chapter I-4 --- Isolation of Valiolamine and Its Related Compounds --- p.6 / Chapter I-5 --- Previous Syntheses of Valiolamine --- p.8 / Chapter II --- Results and Discussions / Chapter II-1 --- General Strategy --- p.17 / Chapter II-2 --- "Synthesis of (lR,2R)-diol (62)" --- p.20 / Chapter II-3 --- Synthesis and Reactivity of Olefin 69 --- p.24 / Chapter II-4 --- "Synthesis of (1R,2S) and (lR,2R)-diastereoisomers 25 and 27" --- p.27 / Chapter II-5 --- "Synthesis of (1S,2R)-diastereoisomer 26 and Valiolamine" --- p.32 / Chapter II-6 --- "Comment on the Regio Chemistry of Nucleophilic Attack of 68, 65 and" --- p.85 / Chapter II-7 --- Results of Biological Test --- p.43 / Chapter III --- Conclusion --- p.46 / Chapter IV --- Experimental --- p.48 / Chapter V --- Reference --- p.80

ABSTRACT: During inflammation, the kynurenine pathway (KP) metabolises the essential amino acid tryptophan (TRP) potentially contributing to excitotoxicity via the release of quinolinic acid (QUIN) and 3-hydroxykynurenine (3HK). Despite the importance of excitotoxicity in the development of secondary brain damage, investigations on the KP in TBI are scarce. In this study, we comprehensively characterised changes in KP activation by measuring numerous metabolites in cerebrospinal fluid (CSF) from TBI patients and assessing the expression of key KP enzymes in brain tissue from TBI victims. Acute QUIN levels were further correlated with outcome scores to explore its prognostic value in TBI recovery. METHODS: Twenty-eight patients with severe TBI (GCS ≤ 8, three patients had initial GCS = 9-10, but rapidly deteriorated to ≤8) were recruited. CSF was collected from admission to day 5 post-injury. TRP, kynurenine (KYN), kynurenic acid (KYNA), QUIN, anthranilic acid (AA) and 3-hydroxyanthranilic acid (3HAA) were measured in CSF. The Glasgow Outcome Scale Extended (GOSE) score was assessed at 6 months post-TBI. Post-mortem brains were obtained from the Australian Neurotrauma Tissue and Fluid Bank and used in qPCR for quantitating expression of KP enzymes (indoleamine 2,3-dioxygenase-1 (IDO1), kynurenase (KYNase), kynurenine amino transferase-II (KAT-II), kynurenine 3-monooxygenase (KMO), 3-hydroxyanthranilic acid oxygenase (3HAO) and quinolinic acid phosphoribosyl transferase (QPRTase) and IDO1 immunohistochemistry. RESULTS: In CSF, KYN, KYNA and QUIN were elevated whereas TRP, AA and 3HAA remained unchanged. The ratios of QUIN:KYN, QUIN:KYNA, KYNA:KYN and 3HAA:AA revealed that QUIN levels were significantly higher than KYN and KYNA, supporting increased neurotoxicity. Amplified IDO1 and KYNase mRNA expression was demonstrated on post-mortem brains, and enhanced IDO1 protein coincided with overt tissue damage. QUIN levels in CSF were significantly higher in patients with unfavourable outcome and inversely correlated with GOSE scores. CONCLUSION: TBI induced a striking activation of the KP pathway with sustained increase of QUIN. The exceeding production of QUIN together with increased IDO1 activation and mRNA expression in brain-injured areas suggests that TBI selectively induces a robust stimulation of the neurotoxic branch of the KP pathway. QUIN's detrimental roles are supported by its association to adverse outcome potentially becoming an early prognostic factor post-TBI.

Quinolinate phosphoribosyltransferase (QPRTase, EC 2.4.2.19) is an intriguing enzyme which appears to catalyse two distinct chemical reactions; transfer of a phosphoribosyl moiety from 5-phosphoribosyl-1-pyrophosphate to the nitrogen of quinolinic acid and decarboxylation at the 2-position to give nicotinic acid mononucleotide. The chemical mechanism of QPRTase is not fully understood. In particular, enzymatic involvement in the decarboxylation step is yet to be conclusively proven. QPRTase is neurologically important as it degrades the potent neurotoxin, quinolinic acid, implicated in diseases such as Huntington’s disease and AIDS related dementia. Due to its neurological importance and unusual chemistry the mechanism of QPRTase is important. Described here is a mechanistic study on human brain QPRTase. Human brain QPRTase was successfully expressed in E. coli BL21 (DE3) from the pEHISTEV-QPRTase construct and the protein was efficiently purified by nickel affinity chromatography. The crystal structure was solved using multiwavelength methods to a resolution of 1.9 Å. Human brain QPRTase was found to adopt an energetically stable hexameric arrangement. The enzyme was also found to exist as a hexamer during gel filtration under physiological conditions. Kinetic studies allowed the measurement of the kinetic parameters for quinolinic acid. The data gave a Km of 13.4 ± 1.0 μM and a Vmax of 0.92 ± 0.01 μM min-1. There was no evidence for cooperative binding of quinolinic acid to the six subunits of the QPRTase hexamer. The enzyme showed maximum activity at approximately pH 6. The active site of human brain QPRTase is a deep pocket with a highly positive electrostatic surface composed of three arginine residues, two lysine residues and one histidine residue. Mutation of these residues resulted in either complete loss or significant reduction in enzymatic activity showing they are important for binding and/or catalysis. A possible mechanism involving QPRTase in the decarboxylation of quinolinic acid mononucleotide was proposed. A series of quinolinic acid analogues were synthesised and tested as inhibitors of QPRTase. The inhibition studies highlighted some key interactions in the active site.

Thesis (M.Sc. (Pharmacy)) - Rhodes University, 2008.

Orientador: Rosângela da Silva de Laurentiz / Resumo: Compostos contendo o núcleo quinolínico apresentam inúmeras propriedades biológicas e estão presentes em muitos fármacos utilizados atualmente contra malária, doenças coronárias e alergias. Existem inúmeras rotas sintéticas para a obtenção de derivados de quinolinas, entretanto, muitas delas são limitadas pelos materiais de partida utilizados e outras não se aplicam, por exemplo, à síntese dos compostos alvos desse trabalho que são lactonas quinolínicas, análogas a lignanas biologicamente ativas. Desta forma, o presente trabalho teve por objetivo propor uma rota sintética mais efetiva para a obtenção de lactonas quinolínicas através do uso de micro-ondas (MO), solventes e reagentes mais adequados reduzindo o tempo de reação e aumentando o rendimento do produto final. Inicialmente foi realizada a síntese dos intermediários anilinolactonas pela reação assistida por micro-ondas entre ácido tetrônico e anilinas. Os produtos foram obtidos após 40 minutos de reação com rendimentos variando de 74 a 98%. Para a obtenção de diferentes lactonas quinolínicas foi realizada a reação entre as várias anilinolactonas e aldeídos aromáticos contendo grupos sacadores e doadores de elétrons. As lactonas quinolínicas foram obtidas com 60 a 97% de rendimento como único produto. Foi testada também a reação para a obtenção de lactonoas dihidroquinolínicas pela reação entre anilinolactonas e aldeídos aromáticos em HCl concentrado. A partir dessa rota foram obtidas as lactonas dihidroquinolínicas com... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Compounds containing the quinoline nucleus have numerous biological properties and are present in many currently used drugs against malaria, coronary diseases and allergies. There are numerous synthetic routes for obtaining quinolines derivatives, however, many of them are limited by the starting materials used and others do not apply, for example to synthesis of the compounds of this work which are quinolinic lactones, analogous to biologically active lignans. The present work propose a more effective synthetic route to obtain quinolinic lactones through the use of microwaves (MW), solvents and reagents more suitable reducing the reaction time and increasing the yield of the final product. Initially the synthesis of the anilinolactones intermediates was carried out by the microwave-assisted reaction between tetronic acid and anilines. The products were obtained after 40 minutes of reaction with yields ranging from 74 to 98%. To obtain different quinolinic lactones the reaction between various anilinolactones and aromatic aldehydes containing electrondonating or electronwithdrawing substituents. The quinolinic lactones were obtained in 60 to 97% yield as the sole product. The reaction for the preparation of dihydroquinolinic lactones by the reaction between anilinolactones and aromatics aldehydes in concentrated HCl was also tested. From this route, the dihydroquinolinic lactones were obtained, as main products, in yields ranging from 55 to 85%. Under these conditions when ... (Complete abstract click electronic access below) / Mestre

La mise au point de médicaments ciblant la neuroinflammation, une composante importante de la physiopathologie des maladies neurodégénératives, fait l’objet de nombreuses recherches. Dans ce travail de thèse, nous avons étudié les effets de deux molécules potentiellement anti-inflammatoires et neuroprotectrices : l’hémine, un inducteur de l’hème oxygénase 1(HO-1) et ; le C16, un inhibiteur de la protéine kinase activée par l’ARN (PKR) dans un modèle de neuroinflammation in vivo par injection intrastriatale d’acide quinolinique (AQ) chez le rat. Nos résultats ont montré que l’induction de la HO-1 produit des effets délétères tandis que l’inhibition de la PKR induit des effets neuroprotecteurs et antiapoptotiques. Ce travail a par ailleurs permis de décrire l’évolution cinétique de la neuroinflammation sur 90 jours dans le modèle AQ, la capacité du tissu cérébral à se régénérer après la lésion et l’intérêt de ce modèle dans l’étude des effets d’agents neuroprotecteurs administrés au long cours. / Neuroinflammation is a key part of the physiopathology of neurodegenerative diseases and is an interesting target in their treatment. In this PhD work, we studied the effects of two potentially anti-inflammatory and neuroprotective molecules, hemin and C16, in an in vivo rat model of neuroinflammation by intrastriatal injection of quinolinic acid (QA). We showed that heme oxygenase 1 (HO-1) induction by hemin has deleterious effects whereas inhibition of the protein kinase RNA activated (PKR) by C16 treatment induced neuroprotective and anti-inflammatory effects. Concurrently, we evaluated longitudinal evolution of neuroinflammation in our model. Results showed the kinetic of the inflammatory phenomena; the ability of cerebral tissue to recover integrity and the capability of this model to evaluate potential neuroprotective and anti-inflammatory drugs in a long-time study.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:19:42Z No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:19:53Z (GMT) No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) / Made available in DSpace on 2016-02-19T13:19:53Z (GMT). No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A malária é uma doença causada por cinco espécies de parasitos do gênero Plasmodium que causa anualmente a morte de milhares de pessoas, principalmente em países pobres da África. Muito antiga, uma diversidade de fármacos já foram empregados na tentativa de erradicação da doença, entretanto o aparecimento de cepas resistentes, bem como efeitos adversos gerados pelo tratamento impossibilitou tal ação. Os quinolínicos configuram uma grande parte destes tratamentos, apresentando uma notável atividade antimalárica. Neste trabalho nós avaliamos o potencial antimalárico de três novos derivados quinolínicos BS 260, BS 318 e BS 373 em culturas de Plasmodium falciparum, cepa w2, cloroquina resistente. BS 373 apresentou melhor atividade contra culturas de Plasmodium falciparum e, assim como o BS 318, foi capaz de inibir a biocristalização de hemozoína pelos parasitos. A microscopia eletrônica de transmissão revelou uma desorganização celular, diminuição do tamanho e quantidade de cristais de hemozoína no vacúolo digestivo, bem como vacuolizações citoplasmáticas e presença de estruturas membranares no vacúolo digestivo, o que indica a ocorrência de um processo autofágico nas células tratadas com 10 LM e 20 LM do BS 373. A presença de cristais citoplasmáticos indica a ocorrência de autólise pela ruptura da membrana do vacúolo digestivo. Por fim, o efeito dos tratamentos se mostrou irreversível nos parasitos com 24 horas de tratamento para BS 318 e BS 373, enquanto que para BS 260 essa irreversibilidade só foi observada após 48 horas. Nossos dados mostram que os derivados quinolínicos testados são efetivos contra culturas de P. falciparum, configurando bons candidatos à novas moléculas antimaláricas. / Malaria is a disease caused by five Plasmodium species that cause deaths of thousands of people annually, mostly in poor countries of Africa. Very anccient, a variety of drugs have been used in an attempt to eradicate the disease, however the emergence of resistant strains, as well as adverse effects caused by treatment prevented such action. The quinoline are a large part of these treatments, presenting a remarkable antimalarial activity. In this paper we evaluate the antimalarial potential of three new quinoline derivative BS 260, BS 318 and BS 373 in Plasmodium falciparum chloroquine resistant, w2 strain, cultures. BS 373 showed the best activity against Plasmodium falciparum cultures, while and analogously to BS 318 was able to inhibit the hemozoin formation by parasites. The transmission electron microscopy revealed a cell disorganization, decreased size and amount of hemozoin crystals in the digestive vacuole, cytoplasmic vacuolization and presence of membrane structures in the digestive vacuole, which indicates an autophagic process in cells treated with 10 LM and 20 LM BS 373. Cytoplasmic being crystals indicate parasite cell autolusis caused by digestive vacuole membrane disrupture. Finally, the effect of treatment proved irreversible on parasites at 24 hours of treatment for BS 318 and BS 373, whereas for BS 260 this irreversibility was only observed after 48 hours. Our data show that the quinoline derivatives tested are effective against P. falciparum cultures, setting good candidates for new antimalarial molecules.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T19:38:00Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CatianeBisogninAlves.pdf: 1685484 bytes, checksum: f55dbdb3d23dcf00e605bf4bcddc7bb7 (MD5) / Made available in DSpace on 2018-08-16T19:38:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CatianeBisogninAlves.pdf: 1685484 bytes, checksum: f55dbdb3d23dcf00e605bf4bcddc7bb7 (MD5) Previous issue date: 2015-03-06 / Statins are cholesterol-lowering agents due to the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Recent studies have shown pleiotropic effects for statins, such as anti-epileptic effect in rodents. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. The nanoparticles have become an important focus of therapeutic research on brain because they are an especially effective form of drug delivery. Therefore, considering the therapeutic potentials of nanocapsules, in the present study the protective effect of simvastatin in nanoencapsulated (SN) or free form (SF) is evaluated in QA-induced seizures in rats. Male adult Wistar rats (250-300 g) were pretreated orally during 21 days with control formulation (drug-unloaded NCs, CF), SN or SF at 1 mg/kg/day. After pretreatment, rats were infused with 4 μL of 239.2 nmol QA at right lateral brain ventricle and observed for behavioral changes during 10 min. Twenty four hours after seizures, rats were evaluated for locomotion, balance, gait and memory. CF had pH 6.75 ± 0.19, particle diameter of 213.91 ± 19.96 nm, polydispersity index of 0.167 ± 0.05 and zeta-potential values were -16,28 ± 7,90. SN had pH 6.68 ± 0.25, particle diameter of 215.25 ± 30.58 nm, polydispersity index of 0.151 ± 0.07 and zeta potential values were -16.13 ± 8.19 mV. The body weight of rats did not change during the treatment period. The pretreatment com SN or SF did not change the seizures or number of death following QA infusion. In the group CF + QA the number of rats with fall in the Beam Balance task was higher than Naive group, effect prevented by SN and SF. The groups CF + Sal and SF + QA showed a reduced number of rearings in the Open Field test, besides the increased stride length in the Footprint test. Rats infused with SF + QA had impairment of memory in the Avoidance Inhibitory Memory task and SN or SF did not change this damage effect. Altogether, the present results exhibit that simvastatin in nanoencapsulated or free form at the dose 1 mg/kg/day administered for 21 days was not able to protect rats against the behavioral damages induced by QA in rats (with exception on balance parameter). Another strategies need to investigate to better evaluate the effect of nanocapsules against cerebral damage induced by QA. / As estatinas são agentes de redução de colesterol devido à inibição da 3-hidroxi-3-metilglutaril coenzima A (HMG-CoA) redutase. Estudos recentes demonstram que as estatinas possuem efeitos pleiotrópicos, como efeito anti-epilético em roedores. Contudo, pequenas quantidades de sinvastatina atravessam a barreira hematoencefálica, sendo necessárias elevadas quantidades do fármaco, gerando os efeitos colaterais indesejáveis, o que poderia ser um problema na sua utilização para o tratamento de doenças que afetam o sistema nervoso central. O ácido quinolínico (AQ) é um análogo endógeno do neurotransmissor glutamato, que está envolvido na etiologia da epilepsia devido a perturbações na liberação e captação de glutamato. As nanopartículas tornaram-se um importante foco de pesquisas terapêuticas para o cérebro, pois é uma forma especialmente eficaz de entrega de fármacos em locais onde há baixa permeabilidade. Portanto, considerando as potencialidades terapêuticas de nanocápsulas, no presente estudo é avaliado o efeito protetor da sinvastatina na forma nanoencapsulada (NS) e livre (SL) no modelo de convulsão induzida por AQ em ratos. Os ratos Wistar adultos machos (250-300 g) foram pré-tratados via oral por 21 dias com nanocápsulas brancas (sem fármaco, NB), NS ou SL, na dose de 1 mg/kg/dia. Após o pré-tratamento, os ratos receberam uma infusão de 4 μL de 239,2 nmol de AQ no ventrículo cerebral lateral direito. Os animais foram observados por 10 minutos para a ocorrência de alterações comportamentais. Vinte e quatro horas após as convulsões, os ratos foram testados quanto locomoção, equilíbrio, marcha e memória. As NB apresentaram um pH 6,75 ± 0,19, diâmetro de partícula de 213,91 ± 19,96 nm, índice de polidispersão de 0,167 ± 0,05 e potencial zeta -16,28 ± 7,90 mV. As NS apresentaram um pH de 6,68 ± 0,25, diâmetro de partícula de 215,25 ± 30,58 nm, índice de polidispersão de 0,151 ± 0,07 e potencial zeta -6,13 ± 8,19mV. Não houve redução do peso corporal dos animais ao longo do tratamento. O pré-tratamento com NS ou SL não causou alteração nas convulsões ou número de mortes ocasionadas pela infusão de AQ. O grupo NB + AQ possui maior número de animais com quedas na tarefa de Equilíbrio na Passarela Elevada, efeito prevenido pelo pré-tratamento com NS ou SL. Os grupos NS + Sal e SL + AQ tiveram uma redução no número de explorações verticais no Campo Aberto, além de apresentarem um aumento no padrão do comprimento da passada no teste de Impressão das Patas. Os ratos tratados com NB + AQ tiveram prejuízo de memória avaliado na tarefa da Esquiva Inibitória, e o pré-tratamento com NS ou SL não preveniu esse efeito. No geral, os resultados demonstram que a sinvastatina nanoencapsulada ou na forma livre na dose de 1 mg/kg/dia administrada por 21 dias não induziu efeitos protetores contra os danos observados após a infusão de AQ (com exceção do equilíbrio). Outras estratégias devem ser exploradas para avaliar o efeito dessas formulações no dano cerebral induzido pelo AQ.

Orientadores: Fabio Rogério, Carlos Amilcar Parada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:39:33Z (GMT). No. of bitstreams: 1 Ignarro_RaffaelaSilvestre_M.pdf: 3644274 bytes, checksum: 6e13f812b2d525e18878656d3ec27815 (MD5) Previous issue date: 2011 / Resumo: A injeção de ácido quinolínico (AQ), um agonista glutamatérgico do receptor N-metil-D-aspartato, no estriado de roedores induz morte seletiva de neurônios espinhosos médios, gliose reativa e neurogênese na zona subventricular, acompanhada da migração dos neurônios recém-gerados para o estriado lesado. Tais achados são também descritos na doença de Huntington (DH). Há indícios de que a via de sinalização JAK/STAT esteja envolvida no mecanismo de ação do AQ, bem como na patogênese da DH. A interação das citocinas da família da IL-6 com seus receptores desencadeia a ativação de enzimas da família das Janus-Quinases (JAKs), que por sua vez permitem o recrutamento e a ativação de fatores de transcrição da família das proteínas transdutoras de sinais e ativadoras da transcrição (STATs). Embora as principais características da DH sejam a presença da coréia e déficits na execução de movimentos voluntários, poucos testes são realizados abordando o comportamento locomotor dos animais no modelo de lesão por AQ. Neste trabalho, estudamos o efeito do AG490, um inibidor da JAK2, na gliose, perda neuronal e neurogênese no estriado de camundongos adultos C57BL/6J após a administração estereotáxica unilateral de AQ (30nmol). Imediatamente após a lesão, os animais receberam uma injeção subcutânea de AG490 (10mg/kg) ou veículo (PBS+DMSO), e injeções diárias por 6 dias adicionais. Além disso, investigamos o possível efeito da lesão por AQ na atividade física voluntária diária (AFVD) em rodas de atividade. A distância percorrida pelos camundongos foi monitorada por 28 dias após a injeção unilateral de QA (30nmol) ou PBS no estriado. Cortes coronais do cérebro (40?m) obtidos em criostato foram utilizados para quantificação de neurônios por estereologia e para a análise de expressão protéica, através de imunoistoquímica e Western Blotting para GFAP e doublecortina, marcadores de gliose e neuroblastos, respectivamente. A área total de células doublecortina-positivas (ACDP) e o número de neurônios (NN) no lado lesado (L) e contralateral à lesão (CL) foram avaliados. O Índice de Neurogênese (IN=ACDP(L)/ACDP(CL)) e o Índice de Sobrevivência Neuronal (ISN=NN(L)/NN(CL)) foram calculados. Após a administração de AQ, o estriado ipsilateral apresentou intensa gliose e células doublecortina positivas com características de células migratórias. O Western Blotting para GFAP mostrou uma redução ipsilateral de 19% nos animais tratados com AG490, em comparação aos animais do grupo tratado apenas com veículo (0.82±0.05; 1.010±0.06, n=9, p<0.05). O ISN foi 25% maior nos camundongos que receberam AG490 em comparação aos animais controles (0.75 ± 0.07; 0.60 ± 0.03; n=8, p<0.05). O IN mostrou uma diminuição de 21% no grupo AG490 em relação ao grupo de animais tratados apenas veículo de diluição (1.08±0.06; 1.37±0.09, n=5, p<0.05). A AFVD média, medida em quilômetros por dia, não se alterou nos animais que receberam injeção intra-estriatal de QA (30nmol) em comparação aos animais do grupo controle (3.97±0.34; 3.90±0.21, n=8, p>0.05). Portanto, nossos resultados suportam um papel para a JAK2 na morte neuronal, gliose, e neurogênese estriatais após lesão com AQ. O tratamento com o inibidor AG490 causou neuroproteção e diminuição da gliose, sugerindo que a reação astrocitária pode prejudicar a sobrevivência neuronal neste modelo experimental / Abstract: Injection of quinolinic acid (QA), a N-methyl-D-aspartate receptor agonist, in murine striatum induces death of medium spiny neurons, gliosis and neurogenesis in the subventricular zone with migration of newly synthesized neurons to damaged striatum. Such findings are also described in Huntington's disease (HD). The Janus-kinase (JAK) pathway would take part in QA mechanism of action and HD pathogenesis as well. The interaction of interleukin-6 family of cytokines with its receptor triggers the activation of enzymes of the family of JAKs, which in turn allow the recruitment and activation of transcription factors, known as signal transducers and activators of transcription (STATs). Although the main features of HD are the presence of chorea and deficits in performing voluntary movements, few tests are realized regarding locomotor behavioral on QA model. We studied the effect of AG490, an inhibitor of JAK isoform 2 (JAK2), on gliosis, neuronal loss and neurogenesis in the striatum of adult C57BL/6J mice after unilateral estereotaxic administration of QA (30 nmol). Immediately after injury, animals received a subcutaneous injection of AG490 (10 mg/kg) or vehicle (PBS + DMSO), and then once daily injections for 6 days. Furthermore, in a parallel experiment, we investigated the possible effect of the lesion by AQ on the voluntary daily physical activity (VDPA) in running wheels. The distance traveled by mice was monitored daily for 28 days after unilateral injection of QA (30 nmol) or PBS into the striatum. Frozen brain sections (40?m) were used for neuronal stereological quantification and immunohistochemical and Western Blotting analyses for GFAP and doublecortin, markers of gliosis and neuroblasts, respectively. The total area of doublecortin-positive cells (ADPC) and the number of neurons (NN) in the lesioned (L) and contralateral (CL) sides were evaluated. Neurogenesis index (NI = ADPC in L/ ADPC in CL) and neuronal survival ratio (NSR = NN in L/ NN in CL) were calculated. After QA administration, ipsilateral striatum showed intense gliosis and doublecortin-positive cells with few processes and ovoid bodies, morphological features corroborating a migratory activity. Western Blotting for GFAP showed an ipsilateral decrease of 19% in AG490- vs vehicle-treated animals (0.82 ± 0.05 vs 1.010 ± 0.06; n=9, p<0.05). NSR was 25% higher in mice given AG490 vs controls given vehicle (0.75 ± 0.07 vs 0.60 ± 0.03; n=8, p<0.05). NI showed a decrease of 21% in AG490- vs vehicle-treated mice (1.08 ± 0.06, 1.37 ± 0.09; n=5, p<0.05). The average VDPA, measured in kilometers per day for 28 days, has not changed in animals that received intrastriatal injection of QA (30nmol) compared to animals that received PBS (3.97 ± 0.34, 3.90 ± 0.21, n = 8, p> 0.05). In conclusion, our results support a role for JAK2 in striatal neuronal death, gliosis and neurogenesis determined by QA. AG490 caused neuroprotection and reduced gliosis suggesting that astrocytic reaction may impair neuronal survival in the present experimental model / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Accumulating evidence suggests that quinolinic acid has a role to play in disorders involving impairment of learning and memory. In the present study, the effect of the guanosine analogue antiherpetic, acyclovir, on quinolinic acid-induced spatial memory deficits was investigated, as well as some of the mechanisms which underlie this effect. Behavioural studies using a Morris water maze show that post-treatment of rats with acyclovir significantly improves spatial memory deficits induced by intrahippocampal injections of quinolinic acid. Histological analysis of the hippocampi show that the effect of acyclovir is related to its ability to alleviate quinolinic acid-induced necrotic cell death, through interference with some of the mechanisms of neurodegeneration. However, acyclovir is unable to alter a quinolinic acid-induced increase in glutamate release in the rat hippocampus, even though it alleviates quinolinic acid induced oxidative stress by scavenging the superoxide anion in vitro and in vivo in whole rat brain and hippocampus respectively. Due to the inverse relationship which exists between superoxide anion and glutathione levels, acyclovir also curtails the quinolinic acid-induced decrease in hippocampal glutathione levels. Acyclovir suppresses quinolinic acid-induced lipid peroxidation in vitro and in vivo, in whole rat brain and hippocampus respectively, through its alleviation of oxidative stress and possibly through the binding of iron (II) and / or iron (III), preventing the participation and redox recycling of iron (II) in the Fenton reaction, which quinolinic acid is thought to enhance by weak binding of ferrous ions. This argument is further strengthened by the ability of the drug to suppress iron (II)-induced lipid peroxidation in vitro directly. Inorganic studies including ultraviolet and visible spectroscopy, electrochemistry and the ferrozine assay show that acyclovir binds to iron (II) and iron (III) and that quinolinic acid forms an easily oxidisable association with iron (II). Acyclovir inhibits the endogenous biosynthesis of quinolinic acid by inhibiting the activity of liver tryptophan-2,3-dioxygenase, intestinal indoleamine-2,3-dioxygenase and rat liver 3-hydroxyanthranillic acid oxygenase in vitro and in vivo, possibly through competitive inhibition of haeme, scavenging of superoxide anion and binding of iron (II) respectively. An inverse relationship exists between tryptophan-2,3-dioxygenase activity and brain serotonin levels. Acyclovir administration in rats induces a rise in forebrain serotonin and 5-hydroxyindole acetic acid and reduces the turnover of forebrain serotonin to 5-hydroxyindole acetic acid. Furthermore, it shows that acyclovir does not alter forebrain norepinephrine levels. The results of the pineal indole metabolism study show that acyclovir increases 5-hydroxytryptophol, N-acetylserotonin and the neurohormone melatonin, but decreases 5-hydroxyindole acetic acid. The results of this study show that acyclovir has some neuroprotective properties which may make it useful in the alleviation of the anomalous neurobiology in neurodegenerative disorders.