Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Ação da β-1,3 glucana no sistema imune de camundongos com sepse por Candida albicans

The frequency of disseminated candidiasis caused by yeast has enhancing in intensive care unit. Despite the availability of new antifungal drugs, C. albicans sepsis mortality causes can be as high as 30-40%. So, it has been needed to looking for a new therapeutic medicament that helps in treatment and prevention of this infection. Previous data that demonstrated that particulated β-glucan stimulates the immune system and experiments of this work were conducted to investigating if β-glucan extracted from Saccharomices cerevisiae, could modified the evolution of mouse model C. albicans systemic infection. Balb/c mice with sepsis and β-1,3 glucan treated or not were analyzed the influence of β-1,3 glucan in survival of the animals, in the fungal burdens in kidney, in the production of urea and TNF even in the histopathology of kidney. The experiments shown that the infected animals a nd glucan treated had great survival (p<0,05), less unit form colony in kidney and normal levels of urea. In the kidney histopathology of not glucan treated animals it has seen more lesions when compared with treated animals. So we conclude that β-1,3 glucan could stimulate the immune system against disseminated C. albicans

Adesão de células mesenquimais da medula óssea de camundongos e do ligamento periodontal de ratos a diferentes superfícies de titânio: estudo comparativo in vitro

The present experiment used cell culture to analyze the adhesion capacity of mouse mesenchymal bone marrow cells and rat periodontal ligament to different titanium surfaces. Grade II ASTM F86 titanium discs 15mm in diameter and 1.5mm thick were used and received 2 distinct surface treatments (polished and cathodic cage plasma nitriding). The cells were isolated from the mouse bone marrow and rat periodontal ligament and cultured in α-MEM basic culture medium containing antibiotics and supplemented with 10% FBS and 5% CO2, for 72 hours at 37ºC in a humidified atmosphere. Subculture cells were cultured in a 24-well plate with a density of 1 x 104 cells per well. The titanium discs were distributed in accordance with the groups, including positive controls without titanium discs. After a 24-hour culture, the cells were counted in a Neubauer chamber. The results show that both the mouse mesenchymal bone marrow cells and rat periodontal ligament cells had better adhesion to the control surface. The number of bone marrow cells adhered to the polished Ti surface was not statistically significant when compared to the same type of cell adhered to the Ti surface treated by cathodic cage plasma nitriding. However a significant difference was found between the control and polished Ti groups. In relation to periodontal ligament cell adhesion, a significant difference was only found between the control and plasma-treated Ti surfaces. When comparing equal surfaces with different cells, no statistically significant difference was observed. We can therefore conclude that titanium is a good material for mesenchymal cell adhesion and that different material surface treatments can influence this process

Avaliação dos efeitos do biperideno, diazepam e neuropeptídeos S sobre a memória e a ansiedade na tarefa do labirinto em T elevado em camundongos

Anxiety is an emotional phenomenon, and normally it is interpreted as an adaptative behavior front to adversities. In its pathological form, anxiety can severely affect aspects related to the personal and professional life. Studies have shown a close relationship between anxiety disorders and aversive memory processing. Considering that the pharmacotherapy of anxiety disorders is still limited, innovative anxiolytic agents are needed. In this regard, neuropeptides systems are interesting therapeutic targets to the treatment of psychopathologies. Neuropeptide S (NPS), a 20-aminoacid peptide, is the endogenous ligand of a G-protein coupled receptor (NPSR), which has been reported to evoke hyperlocomotion, awakefull states, besides anxiolysis and memory improvements in rodents. This study aimed to investigate the effects of biperiden (BPR; an amnesic drug), diazepam (DZP; an anxiolytic drug) and NPS at three distinct times: pre-training, post-training, and pre-test, in order to assess anxiety and memory process in the same animal model. The elevated Tmaze (ETM) is an apparatus derived from the elevated plus-maze test, which consists of one enclosed and two open arms. The procedure is based on the avoidance of open spaces learned during training session, in which mice were exposed to the enclosed arm as many times as needed to stay 300 s. In the test session, memory is assessed by re-exposing the mouse to the enclosed arm and the latency to enter an open arm was recorded. When injected pre-training, BPR (1 mg/kg) impaired learning and memory processing; DZP (1 and 2 mg/kg) evoked anxiolysis, but only at the dose of 2 mg/kg impaired memory; and NPS 0.1 nmol induced anxiolysis without affecting memory. Post-training injection of DZP (2 mg/kg) or BPR (1 and 3 mg/kg) did not affect memory consolidation, while the post-trainning administration of NPS 1 nmol, but not 0.1 nmol, improved memory in mice. Indeed, pre-trainning administration of NPS 1 nmol did not prevent memory impairment elicited by BPR (2 mg/kg, injected before training). In the open field test, BPR 1 mg/kg and NPS 1 nmol induced hyperlocomotion in mice. In conclusion, the proposed ETM task is practical for the detection of the anxiolytic and amnesic effects of drugs. The anxiolytic and memory enhancement effects of NPS were detected in the ETM task, and reinforce the role of NPS system as an interesting therapeutic target to the treatment of anxiety disorders

Estudo dos efeitos comportamentais do neuropeptídeos em camundongos submetidos a modelos animais de Parkinson

Neuropeptide S (NPS) is the endogenous ligand of a G-protein coupled receptor. Preclinical studies have shown that NPSR receptor activation can promote arousal, anxiolytic-like behavioral, decrease in food intake, besides hyperlocomotion, which is a robust but not well understood phenomenon. Previous findings suggest that dopamine transmission plays a crucial role in NPS hyperactivity. Considering the close relationship between dopamine and Parkinson Disease (PD), and also that NPSR receptors are expressed on dopaminergic nuclei in the brain, the current study attempted to investigate the effects of NPS in motor deficits induced by intracerebroventricular (icv) administration of 6-OHDA and systemic administration of haloperidol. Motor deficits induced by 6-OHDA and haloperidol were evaluated on Swiss mice in the rota-rod and catalepsy test. Time on the rotating rod and time spent immobile in the elevated bar were measured respectively in each test. L-Dopa, a classic antiparkinsonian drug, and NPS were administrated in mice submitted to one of the animal models of PD related above. 6-OHDA injection evoked severe motor impairments in rota-rod test, while the cataleptic behavior of 6-OHDA injected mice was largely variable. The administration of L-Dopa (25 mg/kg) and NPS (0,1 and 1 nmol) reversed motor impairments induced by 6-OHDA in the rota-rod. Haloperidolinduced motor deficits on rota-rod and catalepsy tests which were reversed by L-Dopa (100 e 400 mg/kg), but not by NPS (0,1 and 1 nmol) administration. The association of L-Dopa 10 mg/kg and NPS 1 nmol was also unable to counteract haloperidol-induced motor deficits. To summarize, 6-OHDA-, but not haloperidol-, induced motor deficits were reversed by the central administration of NPS. These data suggest that NPS possibly facilitates dopamine release in basal ganglia, what would explain the overcome of motor performance promoted by NPS administration in animals pretreated with 6-OHDA, but not haloperidol. Finally, the presented findings point, for the first time, to the potential of NPSR agonist as an innovative treatment for PD.

Participação do circuito dopaminérgico nas alterações do comportamento de medo inato de camundongos infectados pelo Toxoplasma gondii

The protozoan parasite Toxoplasma gondii transforms the innate aversion of rats for cat urine into a fatal attraction, that increases the likelihood of the parasite completing its life cycle in the cat s intestine. The neural circuits implicated in innate fear, anxiety, and learned fear all overlap considerably, raising the possibility, that T. gondii may disrupt all of these nonspecifically. In this study, we evaluated immunoreactivity for tyrosine hydroxylase (TH) in areas associated with innate fear of infected male swiss mice. The latent Toxoplasma infection converted the aversion of mice to feline odors into attraction. This loss of fear is remarkably specific, as demonstrated by Vyas et al (2007), because infection did not diminish learned fear, anxiety-like behavior, olfaction, or nonaversive learning. However, the neurochemical mechanism related to alterations in innate fear due to T. gondii infection remains poorly studied. 20 mice were inoculated with bradyzoites (25 cysts) from a Toxoplasma gondii (Me-49 strain). The brains were removed after 60 days, sectioned and processed for TH immunohistochemistry. The correlation between the amount of cysts per area and the densitometric analysis of neurotransmitter reactivity was low in the areas implicated in innate fear of infected animals, when comparated with noninfected controls

O Tratamento com a fluoxetina (mas não com outros farmacos antidepressivo e ansioíiticos) reverte o déficit de memória aversiva causado por estresse agudo de contenção em camundongos

The physiologist H. Selye defined stress as the nonspecific response of the body to any factors that endanger homeostasis (balance of internal environment) of the individual. These factors, agents stressors, are able to activate the Hypothalamic-Pituitary-Adrenal (HPA) axis, thus resulting in the physiological responses to stress by the release of glucocorticoids that leads to psychophysiological changes, including effects on cognitive functions such as learning and memory. When this axis is acutely stimulated occurs a repertoire of behavioral and physiological changes can be adaptive to the individual. Notwithstanding, when the HPA axis is chronically stimulated, changes may favor the development of, such as anxiety disorders. Some drugs used in the clinic for the treatment of anxiety disorders these can exert effects on cognitive function, on the HPA axis and on the anxiety. In this context, the aim of our study was to investigate the effects of administration i.p. acute of diazepam (DZP, 2 mg/kg), buspirone (BUS, 3 mg/kg), mirtazapine (MIR, 10 mg/kg) and fluoxetine (FLU, 10 mg/kg) in male mice submitted to acute restraint stress, and evaluated using plus-maze discriminative avoidance task (PMDAT), which simultaneously evaluates parameters such as learning, memory and anxiety. Our results demonstrated that (1) the administration of DZP and BUS, but not FLU, promoted anxiolytic effects in animals; (2) administration mirtazapine caused sedative effect to animals; (3) in the training session, the animals treated with BUS, MIR and FLU learned the task, on the other hand DZP group showed impairment in learning; (4) in the test session, animals treated with DZP, BUS, and MIR showed deficits in relation to discrimination between the enclosed arms, aversive versus non-aversive arm, demonstrating an impairment in memory, however, animals treated with FLU showed no interference in the retrieval of this memory; (5) acute stress did not interfere in locomotor activity, anxiety, or learning on the learning task, but induced impairment in retrieval memory, and the group treated with FLU did not demonstrated this deficit of memory . These results suggest that acute administration of drugs with anxiolytic and antidepressant activity does not interfere with the learning process this aversive task, but impair its retrieval, as well as the acute restraint stress. However, the antidepressant fluoxetine was able to reverse memory deficits promoted by acute stress, which may suggest that modulation, even acutely serotonergic neurotransmission, by selectively inhibiting the reuptake of this neurotransmitter, interferes on the process of retrieval of an aversive memory

Atividade toxicológica do extrato das folhas de Kalanchoe brasiliensis em camundongos Swiss

The species of the genus Kalanchoe (Crassulaceae) are widely used in Brazil as a medicinal product to treat cough, boils, gastritis and other diseases. In this scenario stands out K. brasiliensis, popularly known as coirama or saião. This paper aims at the oral exposure of mice to a hydroalcoholic extract of leaves of Kalanchoe brasiliensis. The animals (total of 100) were divided into 12 groups (6 males and 6 females) for the acute assessment; and groups of 10 for subchronic evaluation. Test groups were treated with doses of 250 mg / kg, 500 mg / kg, 1000 mg / kg and 2000 mg / kg and the control group received 0.9% saline. The animals were observed for 14 days for acute evaluation and 30 days for subchronic evaluation, and in that period analyzed the appearance of clinical signs, changes in weight and consumption of water and food. After the observation period, histopathological analysis of the organs, biochemical serum and haematological parameters for the assessment of the subchronic groups were processed. Differences and changes in body weight were not observed among the groups, nor consumption of water and food, there were no deaths among the groups in the two types of assays. Histopathological analysis showed some alterations compatible with low acute hepatic toxicity. The results of blood glucose, triglycerides, ALT, urea and creatinine showed differences between the control group and the test concentrations studied (p <0.05), but these differences do not show relevant change in the clinical picture of animals. The results showed that the extract K. brasiliensis has low acute toxicity at the doses used and no toxicity when administered for 30 days. This highlights the importance of this species as future and promising candidate for phytotherapics, so has its pharmacological trials completed.

Avaliação dos efeitos do extrato de Passiflora cincinnata Masters em camundongos: efeito na ansiedade e potencial neuroprotetor

Anxiety disorders and Parkinson’s disease (PD) affect a large portion of the world population. Indeed, therapeutic alternatives available do not contribute to improve most clinical conditions and/or are linked with undesirable side effects. Thus, there is a great demand for the development of new drugs to treatment of these diseases. Passiflora cincinnata Mast. is a native species present in several Brazilian states, popularly known as “maracujá do mato”, “maracujá tubarão” or “maracujá mochila”. Additionally, species of Passiflora genus are traditionally known for their exotic flowers, edible fruits with pronounced flavor and for their sedative, tranquilizer and anxiolytic properties reported by folk medicine. These plants possess important organic compounds such as phenols, cyanogenic glycosides, flavonoids and alkaloids, which are responsible for the anxiolytic, antioxidant, anti-inflammatory, antihyperglycemic, among others activities when tested in mammals. Despite this fact, only a few studies have been conducted to investigate the possible in vivo biological effects of Passiflora cincinnata Mast extracts. Thereby, in this study we evaluated the effects of the alcoholic extract of this plant in anxiety and PD animal model. Mice acutely or chronically administered with ethanolic extract of P. cincinnata do not showed any anxiogenic- or anxyolitic-like effect in elevated plus maze (EPM). In order to reproduce PD symptom’s in mice, we administered repeated injections of reserpine which progressively induced motor impairments such as increase in catalepsy, oral movements, and reduction of the average speed of the animals in the open field, as well as depleted dopamine prodution in SNpc cells. Furthermore, this treatment resulted in the loss of aversive memory recall in mice when undergoing PMDAT. Yet, passiflora group also show this amnesic profile. However, animals treated concomitantly with the alcoholic extract of Passiflora cincinnata Mast. showed higher latency for the onset of motor impairment evaluated by catalepsy. Thus, our results shows that the alcoholic extract of the plant P. cincinnata was able to delay the onset of the catalepsy induced by reserpine administration, plus reverted the depletion of dopamine production in SNpc cells.

Efeitos da nicotina no complexo do septo medial na via septo hipocampal em camundongos anestesiados por Ketamina

Nicotine administration in humans and rodents enhances memory and attention, and also has a positive effect in Alzheimer's Disease. The Medial Septum / Diagonal Band of Broca complex (MS/DBB) – a main cholinergic system – massively projects to the hippocampus through the fimbria-fornix, and this pathway is called the septohippocampal pathway. It has been demonstrated that the MS/DBB acts directly on the local field potential (LFP) rhythmic organization of the hippocampus, especially in the rhythmogenesis of Theta (4-8Hz) – an oscillation intrinsically linked to hippocampus mnemonic function. In vitro experiments gave evidence that nicotine applied to the MS/DBB generates a local network Theta rhythm within the MS/DBB. Thus, the present study proposes to elucidate the function of nicotine in the MS/DBB on the septo-hippocampal pathway. In vivo experiments compared the effect of MS/DBB microinfusion of saline (n=5) and nicotine (n=8) on Ketamine/Xylazine anaesthetized mice. We observed power spectrum density in the Gamma range (35 to 55 Hz) increasing in both structures (Wilcoxon Rank-Sum test, p=0.038) but with no change in coherence between these structures in the same range (Wilcoxon Rank-Sum test, p=0.60). There was also a decrease in power of the ketamineinduced Delta oscillation (1 to 3 Hz). We also performed in vitro experiments on the effect of nicotine on membrane voltage and action potential. We patch-clamped 22 neurons in current-clamp mode; 12 neurons were responsive to nicotine, half of them increased firing rate and other 6 decreased, and they significantly differed in action potential threshold (-47.3±0.9 mV vs. -41±1.9 mV, respectively, p=0.007) and halfwidth time (1.6±0.08 ms vs. 2±0.12 ms, respectively, p=0.01). Furthermore, we performed another set of in vitro experiments concerning the connectivity of the three major neuronal populations of MS/DBB that use acetylcholine, GABA or glutamate as neurotransmitter. Paired patch-clamp recordings found that glutamatergic and GABAergic neurons realize intra-septal connections that produce sizable currents in MS/DBB postsynaptic neurons. The probability of connectivity between different neuronal populations gave rise to a MS/DBB topology that was implemented in a realistic model, which corroborates that the network is highly sensitive to the generation of Gamma rhythm. Together, the data available in the full set of experiments suggests that nicotine may act as a cognitive enhancer, by inducing gamma oscillation in the local circuitry of the MS/DBB.

Efeito do ácido ascórbico sobre a espermatogênese de camundongos com distrofia muscular de Duchenne

The objective was to evaluate spermatogenesis alterations caused by DMD and the effect of the treatment using ascorbic acid in preventing those injuries. Twenty four mice were used, 12 from the C57BL/10 lineage (non-dystrophic) and 12 from the C57BL/10Mdx (dystrophic). The sample was divided in six groups containing 4 animals each, as: C30 = 30 days control; D30 = Dystrophic with 30 days; C60 = 60 days control; D60 = Distrophic with 60 days; CS60 = 60 days control supplemented with ascorbic acid and DS60 = Dystrophic with 60 days supplemented with ascorbic acid. The ascorbic acid supplementation was given in the water, 0,005 mg/day. After euthanasia, the testicles (right and left) were collected, weighted and cross sectioned. The material was fixed in the Karnovsky solution for 24 hours, included in resin for histological studies (morphological and morphometric analyzes) submitted to ultrastructural analysis and immunohistochemistry for caspase-3. There was a significant increase in the tunica propria percentage in D30 compared to C30 and D60. The ultrastructural analysis showed mitochondrial apoptosis evidence of Sertoli cells that can reduce sperm efficiency in CS60 and DS60. A higher volume density of apoptotic cells postivas to Caspase-3 in C30 and D30 versus DS60 compared to CS60. There was severe hypertrophy of the Leydig cells between D30 and D60. However, with supplementation was observed reversal of this change in DS60. The ultrastructure of Leydig cells to early presence of lipid vesicles was observed in the group pre-pubertal dystrophic (D30). Thus, the DMD affect the organization of the seminiferous tubules and intertubule, however, the ascorbic acid supplementation used for the treatment of DMD has been just enough to reduce the hypertrophy of the Leydig cells.

Modelagem dos efeitos da irradiação luminosa no cérebro de camundongos e rastreamento de neurônios durante experimentos de microscopia de fluorescência

The fluorescent proteins are an essential tool in many fields of biology, since they allow us to watch the development of structures and dynamic processes of cells in living tissue, with the aid of fluorescence microscopy. Optogenectics is another technique that is currently widely used in Neuroscience. In general, this technique allows to activate/deactivate neurons with the radiation of certain wavelengths on the cells that have ion channels sensitive to light, at the same time that can be used with fluorescent proteins. This dissertation has two main objectives. Initially, we study the interaction of light radiation and mice brain tissue to be applied in optogenetic experiments. In this step, we model absorption and scattering effects using mice brain tissue characteristics and Kubelka-Munk theory, for specific wavelengths, as a function of light penetration depth (distance) within the tissue. Furthermore, we model temperature variations using the finite element method to solve Pennes’ bioheat equation, with the aid of COMSOL Multiphysics Modeling Software 4.4, where we simulate protocols of light stimulation tipically used in optogenetics. Subsequently, we develop some computational algorithms to reduce the exposure of neuron cells to the light radiation necessary for the visualization of their emitted fluorescence. At this stage, we describe the image processing techniques developed to be used in fluorescence microscopy to reduce the exposure of the brain samples to continuous light, which is responsible for fluorochrome excitation. The developed techniques are able to track, in real time, a region of interest (ROI) and replace the fluorescence emitted by the cells by a virtual mask, as a result of the overlay of the tracked ROI and the fluorescence information previously stored, preserving cell location, independently of the time exposure to fluorescent light. In summary, this dissertation intends to investigate and describe the effects of light radiation in brain tissue, within the context of Optogenetics, in addition to providing a computational tool to be used in fluorescence microscopy experiments to reduce image bleaching and photodamage due to the intense exposure of fluorescent cells to light radiation.

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.

Avaliação dos efeitos tóxicos in vitro e in vivo do extrato hidroetanólico dos frutos de Genipa americana L. (Rubiaceae) em camundongos Swiss

The therapeutic use of medicinal plants has contributed since antiquity in a beneficial way for health. However, many species lacks of scientific evidence which provide basis for their use in therapeutic practice. In this context is the Genipa americana L. species (Rubiaceae), popularly known as jenipapo and used to treat syfilis, ulcer and hemorrhagic disturbs. It's also used against bruising, as tonic and as aphrodisiac. Due this species lacks toxicological studies, the aim of this study was to evaluate the toxicity in vivo (acute and sub-chronic toxicity) and in vitro (cytotoxicity) of the hydroethanolic extract from G. americana fruits. The hydroethanolic extract of G. americana fruits was prepared by maceration. A preliminary phytochemical analysis was performed to assess the presence of secondary metabolites in the extract. The cytotoxicity study of the extract (0.1, 1.0, 10, 100 and 1000 mg / 100 ul) were performed against normal cells (3T3) and tumor (786-0, HepG2 and B16), analyzed by the MTT assay. To evaluate the acute (single dose of 2000 mg / Kg) and subchronic (100, 500 and 1000 mg / kg for 30 days) toxicity Swiss mice of both sexes were used. At the end of the experiment, blood samples and organs were collected for analysis. Data between groups were compared by t test or ANOVA with Dunnett's post-test with 5% significance level. The phytochemical study of the extracts mainly indicated the presence of iridoids. Results for cytotoxicity tests showed up to 70% inhibition of B16 cell line at a dose of 1000 mg / 100 ul, and up to 29% inhibition of 786-0 at a dose of 10 ug / 100 ul. The extract did not cause death in 3T3 and HepG2 cells. During the in vivo assays, there were no animal deaths. Analysis of blood samples revealed that the animals submitted to the evaluation of acute toxicity had changes in AST and ALT, and that the animals evaluated for subchronic toxicity showed changes in the relative wet weight of the kidney and plasma urea concentration. No differences were observed between groups on histopathological evaluation of the collected organs. Despite the changes found in the in vivo toxicity tests, using the criteria described by the OECD Guidelines, it is suggested that the hydroethanolic extract of the fruits of the G. americana is classified as low toxicity. The cytotoxicity of the extract suggests that they have potential against melanoma cell lines (B16).