Efeito do T3 reverso sobre o estresse oxidativo induzido pelo hipotireoidismo congênito em hipocampo de ratos imaturos: envolvimento de mecanismos não genômicos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2014.Abstract : Congenital hypothyroidism can lead to a variety of neurochemical and morphophysiological alterations that may be associated with learning deficits, hypomyelination and delayed development of the central nervous system (CNS). In this study, it has been investigated whether the hormone 3,3',5' -triiodothyronine (reverse T3, rT3) is able to reverse the biochemical changes caused by congenital hypothyroidism in hippocampal slices from 15 day-old rats, as well as the mechanism of action of this hormone. Congenital hypothyroidism was induced by adding 0.05% propylthiouracil (PTU) in the drinking water from gestation day 8 and continually up to lactation day 15. Euthyroid rats, receiving only water during the same period, were used as controls. Control and hypothyroid pups were used at 15 day-old, considering the period of maximum synaptogenesis. Hippocampal slices from controls and hypothyroid pups were incubated for 30 min with or without rT3 (10-9 M). Receptor antagonists (RGD and AP-5), inhibitors or activators of cell signaling pathways of p38, CaMKII, ERK1/2, PKA and PKC (SB239063, KN-93, PD98059, H89 and PMA, respectively), as well as L-type voltage-dependent calcium channel blocker (nifedipine) and intracellular calcium chelator (BAPTA -AM), were used to determine the mechanisms involved in the nongenomic action of rT3 on hippocampal cells. We also analyzed some biochemical markers (aminotransferase activity) and oxidative stress parameters, as well as changes in the uptake of 45Ca++ and [14C]-2-deoxy-D-glucose in the hippocampus of immature rats treated or not with rT3. Results showed that hypothyroidism leads to accumulation of intracellular Ca++ and induces oxidative stress. On the other hand, rT3, previously considered as an inactive metabolite of thyroid hormones (TH), was able to reverse, at least some of the biochemical changes induced by congenital hypothyroidism. In this context, rT3 interacts with the surface ?vß3 integrin receptors activating several signal transduction pathways (PKA, CaMKII, ERK1/2 and p38). These events cause a decrease in 45Ca++ influx, stimulation in [14C]-2-deoxy-D-glucose uptake, as well as reverse the oxidative stress induced by congenital hypothyroidism. Considering that changes in TH levels could be associated with neurodegenerative disorders, depression, anxiety, among others, the understanding of mechanisms of action of these hormones might allow the development of more specific and targeted drugs in controlling several physiopathological disorders of CNS

Proposal of semi-nested polimerase chain reaction (PCR) for diagnosing and differentiating BK and JC virus

Objetivos: Implantar e otimizar a reação em cadeia da polimerase (PCR) semi-nested para pesquisa e diferenciação dos vírus BK e JC a partir de amostras clínicas armazenadas. Métodos: Foram testadas 9 amostras clínicas (urina e líquor). As amostras foram submetidas à PCR semi-nested, e foram testadas também com diferentes condições, visando a otimização da reação. Resultados: Primeiramente os controles positivos foram testados quanto à especificidade através de uma PCR direta, e os resultados obtidos confirmaram a especificidade. Quando testadas por PCR semi-nested, as amostras apresentaram os seguintes resultados: 100% (nove amostras) positivas para o vírus BK e 66,6% (seis amostras) positivas para o vírus JC. Conclusões: Otimizou-se a reação de PCR seminested, específica para a identificação dos poliomavírus, para assim atuar como uma ferramenta diagnóstica para melhor acompanhamento de pacientes imunocomprometidos.Aims: To implant and optimize the semi-nested polimerase chain reaction (PCR) to identify and differentiate BK and JC virus. Methods: Nine clinical (urine and liquor) samples have been tested with semi-nested PCR. In order to optimize this reaction, different conditions have been tested. Results: Specificity has been confirmed by using JC and BK positive controls. When tested with semi-nested PCR, the clinical samples showed the following results: 100% (nine samples) positive from BK virus, and 66.6% (six samples) positive from JC virus. Conclusions: The semi-nested PCR reaction, specific for identification of polyomavirus, was optimized for acting as a tool for better follow-up of immunocompromised patients

Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity

AbstractPrevious studies demonstrate that glyphosate exposure is associated with oxidative damage and neurotoxicity. Therefore, the mechanism of glyphosate-induced neurotoxic effects needs to be determined. The aim of this study was to investigate whether Roundup® (a glyphosate-based herbicide) leads to neurotoxicity in hippocampus of immature rats following acute (30min) and chronic (pregnancy and lactation) pesticide exposure. Maternal exposure to pesticide was undertaken by treating dams orally with 1% Roundup® (0.38% glyphosate) during pregnancy and lactation (till 15-day-old). Hippocampal slices from 15 day old rats were acutely exposed to Roundup® (0.00005–0.1%) during 30min and experiments were carried out to determine whether glyphosate affects 45Ca2+ influx and cell viability. Moreover, we investigated the pesticide effects on oxidative stress parameters, 14C-α-methyl-amino-isobutyric acid (14C-MeAIB) accumulation, as well as glutamate uptake, release and metabolism. Results showed that acute exposure to Roundup® (30min) increases 45Ca2+ influx by activating NMDA receptors and voltage-dependent Ca2+ channels, leading to oxidative stress and neural cell death. The mechanisms underlying Roundup®-induced neurotoxicity also involve the activation of CaMKII and ERK. Moreover, acute exposure to Roundup® increased 3H-glutamate released into the synaptic cleft, decreased GSH content and increased the lipoperoxidation, characterizing excitotoxicity and oxidative damage. We also observed that both acute and chronic exposure to Roundup® decreased 3H-glutamate uptake and metabolism, while induced 45Ca2+ uptake and 14C-MeAIB accumulation in immature rat hippocampus. Taken together, these results demonstrated that Roundup® might lead to excessive extracellular glutamate levels and consequently to glutamate excitotoxicity and oxidative stress in rat hippocampus

Efeitos neuroquímicos, histológicos e funcionais do hipotireoidismo congênito e do T3 reverso no sistema nervoso central de ratos em desenvolvimento

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2018.O hipotireoidismo congênito gera uma variedade de alterações neuroquímicas e morfofisiológicas que podem estar relacionadas a déficits cognitivos e falhas no desenvolvimento do sistema nervoso central (SNC). No presente estudo investigou-se as consequências do hipotireoidismo congênito no córtex cerebral e hipocampo de ratos em desenvolvimento, bem como o possível efeito do hormônio 3,3 ,5 -triiodo-L-tironina (T3 reverso; T3r) em reverter as alterações neuroquímicas e morfológicass ocasionadas pela ausência dos hormônios da tireoide (HTs) no hipocampo de ratos de 15 dias de idade. O hipotireoidismo congênito foi induzido com propiltiouracil (PTU) 0,05% na água de beber de ratas Wistar prenhas, desde o 8º dia gestacional até os filhotes completarem 15 dias de idade. O córtex cerebral e o hipocampo de ratos eutireoideos e hipotireoideos foram utilizados para investigar possíveis danos no sistema de glutamatérgico e no sistema redox nessas estruturas cerebrais. O estudo foi dividido em 3 partes: i) e ii) capacidade do T3r em recuperar os danos gerados pelo hipotireoidismo no hipocampo de ratos em desenvolvimento; iii) estudo do efeito do hipotireoidismo congênito no córtex cerebral de ratos em desenvolvimento. Nos estudos i e ii demonstrou-se que os danos neuroquímicos induzidos pelo hipotireoidismo congênito em hipocampo de ratos em desenvolvimento podem ser revertidos, pelo menos em parte, pelo tratamento com T3r ex vivo (1nM por 30 min) ou in vivo (50 ng/Kg por 3 dias consecutivos). O hipotireoidismo levou à alteração no metabolismo do glutamato, bem como, desregulação na fosforilação do citoesqueleto, acúmulo de Ca2+ intracelular, indução do estresse oxidativo e redução da densidade de neurônios NeuN positivos em hipocampo de ratos em desenvolvimento. O T3r foi capaz de reverter estes danos tanto após exposição por 30 minutos quanto por 3 dias consecutivos, demonstrando que esse hormônio atua por mecanismos não genômicos rápidos e sustentados em hipocampo de ratos em desenvolvimento. Desta forma fornecemos evidências que o T3r é um metabolito ativo dos HTs e estes resultados representam uma importante contribuição para elucidar o mecanismo de ação não clássico desse metabólito no SNC em desenvolvimento.Abstract : Congenital hypothyroidism is associated with neurochemical and morphophysiological changes whitin the brain, leading to cognitive deficits and misdevelopment of the central nervous system (CNS). In the present study we investigated the effects of congenital hypothyroidism on the cerebral cortex and hippocampus of developing rats. It was also evaluated the possible effect of the hormone 3,3',5'-triiodo-L-thyronine (reverse T3; rT3) in reversing neurochemical and morphological changes caused by the absence of thyroid hormones (THs) in the hippocampus. Congenital hypothyroidism was induced with 0.05% propylthiouracil (PTU) in the drinking water of pregnant Wistar rats, from the 8th gestational day until the offspring completed 15 days of age. The cerebral cortex and hippocampus of euthyroid and hypothyroid rats were used to investigate possible damage to the glutamatergic system and the redox system of developing rats. The study was divided into three parts: i) and ii) ability of rT3 to restore the hypothyroid-induced cell damage to the hippocampus of developing rats; iii) the histological and neurochemical effects of hypothyroidism in the cerebral cortex of developing rats. Results from studies i and ii have shown that neurochemical damage induced by congenital hypothyroidism in hippocampus of developing rats can be reversed, at least in part, by ex vivo and in vivo treatment with rT3. Hypothyroidism leads to intracellular Ca2+ accumulation, glutamate excitotoxicity, affects cytoskeletal dynamics, decreases the NeuN-positive neuronal density and induces oxidative stress in hippocampus of developing rats. These results were reverted by rT3 through non-genomic and sustained mechanisms in the hippocampus of developing rats. Thus, we provide evidence that rT3 is an active metabolite of THs. These results represent an important contribution to elucidate the non-classical mechanism of action of this metabolite in the developing CNS

Proposta de reação em cadeia da polimerase (PCR) semi-nested para pesquisa e diferenciação dos vírus BK e JC = Proposal of semi-nested polimerase chain reaction (PCR) for diagnosing and differentiating BK and JC virus

Objetivos: Implantar e otimizar a reação em cadeia da polimerase (PCR) semi-nested para pesquisa e diferenciação dos vírus BK e JC a partir de amostras clínicas armazenadas. Métodos: Foram testadas 9 amostras clínicas (urina e líquor). As amostras foram submetidas à PCR semi-nested, e foram testadas também com diferentes condições, visando a otimização da reação. Resultados: Primeiramente os controles positivos foram testados quanto à especificidade através de uma PCR direta, e os resultados obtidos confirmaram a especificidade. Quando testadas por PCR semi-nested, as amostras apresentaram os seguintes resultados: 100% (nove amostras) positivas para o vírus BK e 66,6% (seis amostras) positivas para o vírus JC. Conclusões: Otimizou-se a reação de PCR seminested, específica para a identificação dos poliomavírus, para assim atuar como uma ferramenta diagnóstica para melhor acompanhamento de pacientes imunocomprometido

Proposal of semi-nested polimerase chain reaction (PCR) for diagnosing and differentiating BK and JC virus

Objetivos: Implantar e otimizar a reação em cadeia da polimerase (PCR) semi-nested para pesquisa e diferenciação dos vírus BK e JC a partir de amostras clínicas armazenadas. Métodos: Foram testadas 9 amostras clínicas (urina e líquor). As amostras foram submetidas à PCR semi-nested, e foram testadas também com diferentes condições, visando a otimização da reação. Resultados: Primeiramente os controles positivos foram testados quanto à especificidade através de uma PCR direta, e os resultados obtidos confirmaram a especificidade. Quando testadas por PCR semi-nested, as amostras apresentaram os seguintes resultados: 100% (nove amostras) positivas para o vírus BK e 66,6% (seis amostras) positivas para o vírus JC. Conclusões: Otimizou-se a reação de PCR seminested, específica para a identificação dos poliomavírus, para assim atuar como uma ferramenta diagnóstica para melhor acompanhamento de pacientes imunocomprometidos.Aims: To implant and optimize the semi-nested polimerase chain reaction (PCR) to identify and differentiate BK and JC virus. Methods: Nine clinical (urine and liquor) samples have been tested with semi-nested PCR. In order to optimize this reaction, different conditions have been tested. Results: Specificity has been confirmed by using JC and BK positive controls. When tested with semi-nested PCR, the clinical samples showed the following results: 100% (nine samples) positive from BK virus, and 66.6% (six samples) positive from JC virus. Conclusions: The semi-nested PCR reaction, specific for identification of polyomavirus, was optimized for acting as a tool for better follow-up of immunocompromised patients