Sinalização endógena do sistema Nociceptina/Orfanina FQ - receptor NOP: envolvimento na modulação da depressão experimental induzida por lipopolissacarídeo

During the last decades, it has been established that there is a relationship between major depression and activation of immune system. Nociceptin/orphanin FQ (N/OFQ) is the natural ligand of a Gi-protein coupled receptor named NOP, both compose the peptidergic system wich is involved in the regulation of mood states and inflammatory responses. Considering these actions, the present thesis aimed to investigate the consequences of blocking NOP signaling in lipopolysaccharide (LPS)-induced sickness and depressive-like behaviors in mice. Systemic administration of LPS doses, that do not cause sepsis in mice, induce changes in their behaviors related with activity of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukins 6 (IL-6) and 1β (IL-1 β). At the time points of 2 to 6 h and 24 h after intraperitoneal injection, mice treated with LPS displayed, respectively, sickness and depressive-like behaviors. In the present work the administration of LPS 0.8 mg/kg (ip) significantly induced sickness signs in Swiss and CD-1 mice, such as weight loss, transient reduction in rectal temperature and decrease of food and water intake. Moreover at 24 h after LPS injection these same mice strains displayed significantly increased immobility time on the tail suspension test (TST) when compared with control mice, this alteration was not related with possible locomotion impairments as verified on the open field test. Treatment with Nortriptyline 30 mg/kg (ip, 60 min prior the TST) reduced the immobility time of control and LPS-treated mice and was used as standard antidepressant. The NOP receptor antagonist SB-612111 (10 mg/kg, ip), 30 min prior LPS, did not modify LPS-induced sickness signs and depressive-like behavior. However, when injected 24 h after LPS treatment, SB-612111 (ip, 30 min prior the TST) as well as the peptidergic NOP receptor antagonist UFP-101 (10 nmol/2μL, icv, 5 min prior the TST) significantly reversed the toxin effects. The protocol of LPS-induced depressive-like states was also tested in NOP receptor knockout mice (NOP(-/-)) and their respective wild types (NOP(+/+)). LPS evoked transient rectal temperature reduction in NOP(-/-) mice and loss of body weight, food and water intake reduction in both NOP(+/+) and NOP(-/-) mice. The consumption of water was significantly different due to the genotype. LPS injection induced transient changes in pro-inflammatory cytokines. At 6 h after LPS injection, serum levels of TNF-α were significantly increased in NOP(+/+) and NOP(-/-) mice, as the IL-6 levels were significantly increased just in NOP(+/+) serum. At 24 h after LPS treatment the pro-inflammatory cytokines had returned to the baseline levels in both genotypes. LPS treatment elicited depressive-like effects in NOP(+/+) but not in NOP(-/-) mice. The data obtained during the execution of this doctoral thesis reveal that pharmacological and genetic blockade of NOP signaling does not affect LPS evoked sickness signs while reversing depressive-like behavior. In conclusion, these results highlight the involvement of the peptidergic system N/OFQ - NOP receptor in the modulation of behaviors related to mood and activation of the immune system.

Mapeamento das projeções retinianas para o sistema óptico acessório, colículo superior e complexo pré-tectal do sagui (callithrix jacchus)

The accessory optical system, the pretectal complex, and superior colliculus are important control centers in a variety of eye movement, being extremely necessary for image formation, consequently to visual perception. The accessory optical system is constituted by the nuclei: dorsal terminal nucleus, lateral terminal nucleus, medial terminal nucleus and interstitial nucleus of the posterior superior fasciculus. From a functional point of view they contribute to the image stabilization, participating in the visuomotor activity where all system cells respond to slow eye movements and visual stimuli, which is important for the proper functioning of other visual systems. The pretectal complex comprises a group of nuclei situated in mesodiencephalic transition, they are: anterior pretectal nucleus, posterior pretectal nucleus, medial pretectal nucleus, olivary pretectal nucleus and the nucleus of the optic tract, all retinal projection recipients and functionally are related to the route of the pupillary light reflex and the optokinetic nystagmus. The superior colliculus is an important subcortical visual station formed by layers and has an important functional role in the control of eye movements and head in response to multisensory stimuli. Our aim was to make a mapping of retinal projections that focus on accessory optical system, the nuclei of pretectal complex and the superior colliculus, searching mainly for pretectal complex, better delineation of these structures through the anterograde tracing with the B subunit of cholera toxin (CTb) followed by immunohistochemistry and characterized (measured diameter) synaptic buttons present on the fibers / terminals of the nucleus complex pré-tectal. In our results accessory optical system, including a region which appears to be medial terminal nucleus and superior colliculus, were strongly marked by fibers / terminals immunoreactive CTb as well as pretectal complex in the nucleus: optic tract, olivary pretectal nucleus, anterior pretectal nucleus and posterior pretectal nucleus. According to the characterization of the buttons it was possible to make a better definition of these nucleus.

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Redução dos níveis de Ocratoxina A por ação da enzima peroxidase

A ocratoxina A é um composto formado a partir do metabolismo secundário de fungos dos gêneros Aspergillus e Penicillium. Uma vez que a presença dessa micotoxina nos alimentos causa sérios danos à saúde humana e animal, surge o interesse pelo desenvolvimento de métodos que visem a redução dos seus níveis em diferentes matrizes. Diversos processos de descontaminação têm sido propostos, sendo que os métodos de redução biológica tem recebido destaque. Esses métodos consistem na aplicação de micro-organismos ou de suas enzimas, o que gera a biotransformação ou degradação da toxina produzindo metabólitos com menor ou nenhuma toxicidade. Diante disso, o objetivo geral do trabalho foi avaliar o efeito da peroxidase na redução dos níveis de ocratoxina A. As enzimas peroxidases testadas foram a comercial e a obtida do farelo de arroz. Para a extração enzimática foram utilizadas as frações granulométricas do farelo de arroz de 48 a 100 mesh, sendo estas frações caracterizadas quimicamente. A peroxidase foi extraída do farelo de arroz em tampão 10 mM pH 5,0 e purificada por partição trifásica, obtendo 77,1% de recuperação e 9,2 para o fator de purificação. O método utilizado para a extração da ocratoxina A do sistema aquoso foi por partição líquido-líquido utilizando como solvente o clorofórmio, sendo esse método validado segundo os parâmetros de linearidade (0,1 a 20 ng mL-1), coeficientes de correlação (0,9997) e de determinação (0,9994), e limites de detecção (0,02) e quantificação (0,03). A afinidade entre as peroxidases e a ocratoxina A foi verificada segundo os parâmetros de KM e Vmáx, resultando em 0,00027 mM e 0,000015 mM min-1, respectivamente, para a peroxidase comercial, e 0,0065 mM e 0,000031 mM min-1 para a obtida do farelo de arroz. Com relação aos percentuais de redução de ocratoxina A, foram avaliadas 3 proporções enzima:substrato (1:10, 1:5 e 8:1 para a comercial e 1:10, 1:5 e a com atividade de 0,063 U mL-1 para a do farelo), sendo que as proporções que forneceram maior redução foi a de 8:1 para a enzima comercial (0,063 U mL-1) e a correspondente a 0,063 U mL-1 para a enzima obtida do farelo. Os percentuais de redução de ocratoxina A foram de 59% para a peroxidase comercial em 300 min e 41% para a peroxidase do farelo de arroz em 1440 min. O efeito de adsorção da ocratoxina A pela enzima peroxidase foi descartado uma vez que foi realizada a sua hidrólise com a enzima pepsina e verificado um percentual de 2,7% de adsorção, demonstrando que a redução foi por ação enzimática. A enzima obtida de farelo de arroz com atividade de 0,063 U mL-1 foi aplicada em suco de uva tinto e branco. Observou-se que para o primeiro não houve redução significativa, enquanto que para o segundo a redução foi de 17%. Neste trabalho, então, foi possível verificar a capacidade de redução dos níveis da ocratoxina A pela enzima peroxidase, tanto em sistema aquoso como no suco de uva integral branco.

Interação da cianotoxina microcistina e o nanomaterial de carbono fulereno (C60) em brânquias do peixe Cyprinus carpio (Teleostei: Cyprinidae) sob incidência de radiação UVA

A produção mundial de nanomateriais tem aumentado nos últimos anos, em função de suas variadas aplicações tecnológicas e, como consequência do seu crescente uso e demanda, poderão existir riscos ambientais sendo a água o ambiente onde muitas destas substâncias podem exercer efeitos deletérios. Um dos nanomaterias de carbono mais utilizados é o fulereno, um composto orgânico lipofílico que pode se comportar como carreador de moléculas tóxicas, potencializando a entrada de contaminantes ambientais em órgãos específicos, fenômeno conhecido como “cavalo de Troia”. As microcistinas (MC) são cianotoxinas produzidas por cianobactérias durante episódios de floração, afetando aos organismos aquáticos e ao ser humano. Diversos estudos demonstram que organismos expostos tanto às MCs quanto ao fulereno podem causar produção excessiva de espécies ativas de oxigênio e alterar os níveis de antioxidantes. Além disso, outro fator que pode vir a intensificar o potencial tóxico de ambos é a incidência de radiação UVA. Sendo assim, procurou-se avaliar os efeitos em parâmetros de estresse oxidativo da co-exposição ex vivo da cianotoxina microcistina-LR (MC-LR) e o nanomaterial de carbono fulereno em brânquias do peixe Cyprinus carpio sob incidência de radiação UVA. Os resultados mostraram que: (a) houve uma perda da capacidade antioxidante no tratamento com MC-LR (baixa concentração) quando coexposta com fulereno no UVA em relação com o tratamento realizado sem co-exposição com fulereno; (b) o fulereno no UV diminuiu a atividade da enzima glutationa-Stransferase (GST) quando comparado com o controle no UV; (c) a MC-LR (alta concentração) co-exposta com fulereno foi capaz de diminuir as concentrações do antioxidante glutationa (GSH) quando comparado com o mesmo tratamento tanto no UVA quanto no escuro sem a co-exposição ao fulereno; (d) o tratamento MC-LR (baixa concentração) com UVA aumentou o dano oxidativo lipídico quando comparado com o controle UVA; (e) o fulereno não causou uma maior bioacumulação da microcistina no tecido. Sendo assim, pode-se concluir que o fulereno não apresentou o potencial de carregador de moléculas nessas concentrações de microcistina, porém, a co-exposição dos compostos diminuem tanto capacidade antioxidante total, como a concentração da GSH, podendo gerar problemas a longo prazo na detoxificação da toxina.

Sorological evaluation of commercial vaccines against enterotoxemia in goats.

Enterotoxemia in sheep and in goats is caused by the effects of the epsilon toxin of C/ostridium perfringens type D, being considered the main infectious cause of mortality in those animal species. The main prophylactic measures include adequate nutritional management and vaccination of ali animais using vaccines of high immunogenic power. Six commercial vaccines containing in its formulation the epsilon toxoid of C. perfringens type D were sorogically evaluated. Eighty four female goat kids, whose mothers had no previous vaccination history against clostridioses were used. They were divided into six groups of 14 animais each. The animais of the control group didn't receive any vaccine dose and the animais from the groups 1 to 5 received two vaccine doses, The first vaccine dose was applied at 45 days of life (day zero) and the second dose at 75 days (30 days after the first dose). Blood samples were collected from the goat kids at the days zero, 30, 60, 90, 120 and 150 after the beginning of the experiment, in order to evaluate the immunologic response. The Indirect ELlSA technique was used for the quantification of the antibodies against epsilon toxin in the samples of blood serum of the animais. In day zero, no animal presented titre considered protector. The largest number of animais considered protected was found at day 60, in response to the two initial doses of the vaccine (days O and 30, first and second doses, respectively). Only tive animaIs which received the vaccine 1 and one animal which received the vaccine 3 stayed wilh titres of antibodies considered up to 150 days after the first vaccine dose. Based on the results, it was concluded lhat the evaluated vaccines showed small amount of epsilon toxoid in the commercial formulations, a crucial fact for lhe low efficiency of. the vaccines. For commercial reasons, the vaccines against the clostridioses present versatile formulations, with several toxoid types, used for various animal species, which certainly contributed to reduce their effectiveness in preventing the iIInesses caused by the clostridia or their toxins.

Relationship between super antigenicity, antimicrobial resistance and origin of Staphylococcus aureus isolated

Introduction: Staphylococcus aureus is a pathogen that causes food poisoning as well as hospital and community acquired infections. Objective: Establish the profile of superantigen genes among hospital isolates in relation to clinical specimen type, susceptibility to antibiotics and hospital or community acquisition. Methods: Eighty one isolates obtained from patients at Colombian hospital, were classified by antimicrobial susceptibility, specimen type and hospital or community acquired . The PCR uniplex and multiplex was used for detection of 22 superantigen genes (18 enterotoxins, tsst-1 and three exfoliative toxins). Results: Ninety five point one percent of isolates harbored one or more of the genes with an average of 5.6 genes. Prevalence of individual genes was variable and the most prevalent was seg (51.9%). Thirty nine genotypes were obtained, and the genotype gimnou (complete egc cluster) was the most prevalent alone (16.0%) and in association with other genes (13.6%). The correlation between presence of superantigens and clinical specimen or antimicrobial susceptibility showed no significant difference. But there was significant difference between presence of superantigens and the origin of the isolates, hospital or community acquired (p= 0.049). Conclusions: The results show the variability of the superantigen genes profile in hospital isolates and shows no conclusive relationship with the clinical sample type and antimicrobial susceptibility, but there was correlation with community and hospital isolates. The analysis of the interplay between virulence, epidemic and antibiotic resistance of bacterial populations is needed to predict the future of infectious diseases.