Caracterização do perfil do ciclo sono-vigília em ratos sob dessincronização forçada

The circadian behavior associated with the 24 hours light-dark (LD) cycle (T24) is due to a circadian clock , which in mammals is located in the hypothalamic suprachiasmatic nucleus (SCN). Under experimental conditions in which rats are espoused to a symmetric LD 22h cycle (T22) the two SCN regions, ventrolateral (vl) and dorsomedial (dm), can be functionally isolated, suggesting that each region regulates distinct physiological and behavioral components. The vl region regulates the locomotor activity and slow wave sleep (SWS) rhythms, while the dm region assures the body temperature and paradoxical sleep (PS) rhythms regulation. This research aimed to deepen the knowledge on the functional properties of circadian rhythmicity, specifically about the internal desynchronization process, and its consequences to locomotor activity and body temperature rhythms as well as to the sleep-wake cycle pattern in rats. We applied infrared motion sensors, implanted body temperature sensors and a telemetry system to record electrocorticogram (ECoG) and electromyogram (EMG) in two rat groups. The control group under 24h period LD cycle (T24: 12hL-12hD) to the baseline record and the experimental group under 22h period LD cycle (T22: 11hL- 11hD), in which is known to occur the uncoupling process of the circadian locomotor activity rhythm where the animals show two distinct locomotor activity rhythms: one synchronized to the external LD cycle, and another expressed in free running course, with period greater than 24h. As a result of 22h cycles, characteristic locomotor activity moment appear, that are coincidence moments (T22C) and non coincidence moments (T22NC) which were the main focus or our study. Our results show an increase in locomotor activity, especially in coincidence moments, and the inversion of locomotor activity, body temperature, and sleep-wake cycle patterns in non coincidence moments. We can also observe the increase in SWS and decrease in PS, both in coincidence and non coincidence moments. Probably the increases in locomotor activity as a way to promote the coupling between circadian oscillators generate an increased homeostatic pressure and thus increase SWS, promoting the decreasing in PS

Estimulação optogenética do septo medial no rato anestesiado e em livre comportamento

Theta rhythm consists of an electrophysiological hippocampal oscillation present in mammalian species (4-12 Hz with variations across species). This oscillation is present during active waking and is also prevalent in local field potentials (LFP) during rapid eye movement sleep (REM sleep). Several studies have shown that theta rhythm is important in cognitive tasks and that the medial septum is a key region for its occurrence. The septum sends cholinergic, GABAergic and glutamatergic projections to the hippocampus, which in turn projects axons to the septum. Besides the septum, other regions are involved in regulating theta rhythm, forming a complex network of interactions among brain areas that result in theta rhythm. Optogenetics is a recently developed method that has been widely used in various research areas. It allows us to manipulate the electrical activity of neurons through light stimulation. One of the existing techniques consists in using a viral vector to induce the neuronal expression of ion channels associated with the light-sensitive molecule rhodopsin (e.g. ChR2). Once infected, the neurons become sensitive to light of a particular wavelength. The present M. Sc. research aimed to perform luminous stimulation of the brain in anesthetized and freely behaving animals using chronically implanted electrodes and optical fibers in animals infected with a viral vector for ChR2 expression. Surgical viral injections were performed in the medial septum; histological results confirmed the expression of ChR2 by way of the presence of the eYFP reporter protein in the septum and also in hippocampal processes. Moreover, we performed acute experiments with luminous stimulation of the medial septum and LFP recordings of the septum and hippocampus of anesthetized animals. Action potentials were recorded in the septum. In these experiments we observed a significant increase in the firing rates of septal neurons during luminous stimulation (n = 300 trials). Furthermore, we found an early light-evoked response in the hippocampal LFP. Chronic experiments with luminous stimulation of the medial septum and hippocampus in freely behaving animals were also performed in combination with LFP recordings. We found that the luminous stimulation of the septum is able to induce theta rhythm in the hippocampus. Together, the results demonstrate that the luminous stimulation of the medial septum in optogenetically-modified animals causes relevant electrophysiological changes in the septum and the hippocampus.

Efeitos da senescência no folheto intergeniculado do tálamo de ratos: análises morfológicas e neuroquímicas

The circadian timing system (CTS), in rodents, consists of interconnected neural structures such as the suprachiasmatic nucleus (SCN) of the hypothalamus, Intergeniculate Leaflet (IGL) of the thalamus, synchronous pathways and behavioral effectors. The SCN has been described as the major circadian pacemaker in several species of mammals, while the IGL appears to be involved in integration of photic and non-photic clues relaying them to SCN. The CTS allows an ordered internal temporal organization to the organism, providing the proper execution of physiological and behavioral mechanisms, which brings homeostasis. However, this stability is disrupted with aging process causing numerous pathological disorders, ranging from simple loss of physiological functions to decreases in cognitive performance. Therefore, is fundamental understanding the effects of senescence in this system. In this context, is proposed in this study to check if there are changes in IGL cytoarchitecture, neurochemical and retinal afferent markers with aging and their possible morpho-functional implications. To achieve this goal wistar rats were divided into 3 groups: young (3 months); Middle Age (13 months); Old (23 months). They were submitted to paraformaldhyde (4%) transcardiac perfusion to tissue fixation. Then, they had their brain removed and sectioned in 30 µm slices, which every sixth section were collected. This sections were processed by nissl method and immunostaining for GFAP, GAD, ENK, NPY and CTb in order to analyze the IGL features. It was observed a cell loss in middle age and old animals at Nissl, NPY and CTb stains. In addition, it was shown a increase in GFAP in middle aged animals compared to young and old ones. No differences were found in other neurochemichal stains. These data suggests IGL loss retinal afferents and neurons, in special the NPY-IR ones, likely having a compensatory gliogenesis. This supports the correlations between the CTS functional deficits and an anatomical deterioration of its components with the aging process.

Bioquímica quântica na diferenciação dos níveis de ativação de receptores AMPA por agonistas parciais Wilardina

In the central nervous system (CNS) of mammalian, fast synaptic transmission between nerve cells is performed primarily by α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors, an ionotropic glutamate receptor that is related with learning, memory and homeostasis of the nervous system. Impairments in their functions are correlated with development of many brain desorders, such as epilepsy, schizophrenia, autism, Parkinson and Alzheimer. The use of willardiine analogs has been shown a powerful tool to understanding of activation and desensitization mechanisms of this receptors, because the modification of a single ligand atom allows the observation of varying levels of efficacy. In this work, taking advantage of Fluorine Willardiine (1.35Å), Hydrogen Willardiine (1.65Å), Bromine Willardiine (1.8Å) and Iodine Willardiine (2.15Å) structures co-crystalized with GluA2 with codes 1MQI, 1MQJ, 1MQH and 1MQG, we attempted to energetically differentiate the four ligands efficacy. The complexes were submitted to energetic calculations based on density functional theory (DFT), under the optics of molecular fractionation with conjugate caps (MFCC) method. Obtained results show a relationship between the energetic values and willardiines efficacy order (FW> HW > BrW > IW), also show the importance of E705, R485, Y450, S654, T655, T480 e P478 as the amino acids that contribute most strongly with the interaction of four partial agonists. Furthermore, we outlined the M708 behaviour, attracted by FW and HW ligands, and repels by BrW and IW. With the datas reported on this work, it is possible for a better understanding of the AMPA receptor, which can serve as an aid in the development of new drugs for this system.

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.

Citoarquitetura e imunoistoquímica para tirosinahidroxilase dos núcleos dopaminérgicos mesencefálicos do morcego (Artibeus planirostris)

The 3-Hidroxytyramine/dopamine (DA) is a monoamine of catecholamine family and isthe precursor substance synthesis of noradrenaline and adrenaline, having the enzymeTyrosine Hydroxylase (TH) as this regulatory process. In addition, the DA has theability to act as a neurotransmitter in the Central Nervous System - SNC, being themain neurotransmitter of brain nuclei, namely of A8 to A16. The nuclei of the midbrainthat express DA are the Retrorubral Field (RRF, A8), the Substantia Nigra parsCompacta (SNc, A9) and the Ventral Tegmental Area (VTA, A10). Such nuclei areinvolved in complex three circuitry that are the mesostriatal, mesolimbic andmesocotical and are directly related with several behavioral manifestations as motricitycontrol, reward signaling in behavioral learning, motivation and pathologicalconditions, such as Parkinson's Disease and schizophrenia. Interestingly, many of themorphological bases of these neural disturbance remain unknown. Considering therelevance of mesencephalic dopaminergic nuclei, the aim of this research is tocharacterize morphologically the dopaminergic nuclei (clusters A8, A9 and A10) of themidbrain of the bat (Artibeus planirostris). The Artibeus planirostris is a common bat inRio Grande do Norte. Ten animals were used in this research. The animals wereanesthetized, perfused, and the brain was removed from the skull. After dehydration insucrose, the brain was underwent microtomy. Saggital and coronal sections wereobtained and collected in six separate series. The series were Nissl-stained to identifythe cytoarchitectonic boundaries and the other series were subjected toimmunohistochemistry for TH. After cytoarchitectonic analysis and TH+ cellsidentification was possible to establish the anatomical boundaries of the nuclei, as wellas the subdivisions of three of the midbrain dopaminergic nuclei. The SNc is the mostrostral nucleus observed in the midbrain and is identified throughout the rostrocaudalextension of the midbrain. The VTA neurons were seen immediately caudal to the SNcappearance. The RRF neurons were observed just in the caudal levels of the midbrain.The SNc in Artibeus planirostris shows a particular feature, the tail of the SNc. The tailhave been described just in two other studied species. The present work shows aparticular variation in the organizational morphology of the SNc in the artibeus andcontribute to understand the phylogenetic routes by which the dopaminergic system hasevolved.

Gato-do-mato-pequeno (Leopardus tigrinus) na Caatinga: ocupação e padrão de atividade de um felídeo ameaçado e pouco conhecido na floresta tropical seca do nordeste do Brasil

While the carnivores are considered regulators and structuring of natural communities are also extremely threatened by human activities. Endangered little-spotted-cat (Leopardus tigrinus) is one of the lesser known species from the Neotropical cats. In this work we investigate the occupancy and the activity pattern of L. tigrinus in Caatinga of Rio Grande do Norte testing: 1) how environmental and anthropogenic factors influence their occupation and 2) how biotic and abiotic factors influence their activity pattern. For this we raised occurrence data of species in 10 priority areas for conservation. We built hierarchical models of occupancy based on maximum likelihood to represent biological hypotheses which were ranked using the Akaike Information Criterion (AIC). According to the results the feline occupancy is more likely away from rural settlements and in areas with a higher proportion of woody vegetation. The opportunistic killing of L. tigrinus and in retaliation for poultry predation close to residential areas can explain this result; as well as more complex vegetation structure can better serve as refuge and ensure more food. Analyzing the records of the species through circular statistics we conclude that the activity pattern is mostly nocturnal, although considerable crepuscular and a small diurnal activity. L. tigrinus activity was directly affected by the availability of small terrestrial mammals, which are essentially nocturnal. In addition, the temperatures recorded in the environment directly and indirectly affect the activity of the little-spotted-cat, as also influence the activity of their potential prey. Generally, the cats were more active when possible prey were active, and this happened at night when lower temperatures are recorded. Moreover, the different lunar phases did not affect the activity pattern. The results improve the understanding of an endangered feline inhabiting the Caatinga biome, and thus can help develop conservation and management strategies, as well as in planning future research in this semi-arid ecosystem.

The song of humpback whale and the potential effects of whale-watching in the abrolhos bank, BA, Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The song of humpback whale and the potential effects of whale-watching in the abrolhos bank, BA, Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Áreas importantes para a conservação do último grande herbívoro da caatinga potiguar: o veado-catingueiro (Mazama gouazoubira)

Cervídeo Mazama gouazoubira é a espécie de veado que possui maior distribuição dentre as espécies sul americanas. Aspectos da vegetação, predação, competição, caça e perda de habitat são fatores importantes para compreender a distribuição espacial de herbívoros na paisagem. Dessa forma, considerando a falta de estudos de mamíferos de médio e grande porte na Caatinga, objetivamos compreender quais os fatores ambientais e antrópicos que influenciam a ocupação do veado-catingueiro (M. gouazoubira) na região semiárida do estado do Rio Grande do Norte, Brasil. O estudo foi realizado em 10 áreas amostrais e utilizamos armadilhas fotográficas para amostragem e modelos de ocupação foram construídos representando nossas hipóteses biológicas. Os modelos foram ranqueados através dos valores obtidos do AIC. As variáveis cobertura vegetal de caatinga e densidade da vegetação mostraram maior efeito, já a caatinga arbórea mostrou um efeito menor, sugerindo que a espécie é altamente dependente da vegetação para ocorrer, possuindo menor dependência de caatinga arbórea. Esperamos que nossos resultados possam ser utilizados como suporte para planos de manejo e criação de novas unidades de conservação no estado.

Encalhes de Cetáceos ocorridos no período de 1984 a 2005 no litoral do Rio Grande do Norte, Brasil

Strandings of live or dead aquatic mammals constitute an important instrument to provide information regarding the occurrence, biology and ecology of these species. The aim of this study was to register the stranded species of cetaceans, the frequency and the spatial-temporal distribution of theses strandings during the period of 1984 to 2005, in the coast of Rio Grande do Norte. Data was acquired through the monitoring of strandings in the north, north-west and south coast of RN, and through information obtained from institutions and newspaper archives of the State. A total of 122 strandings of cetaceans were registered along the coast of Rio Grande do Norte. Of the 14 species of cetaceans registered, four species had higher frequencies: Sotalia guianensis (n= 65), Steno bredanensis (n = 6), Globicephala macrorhynchus (n = 6) and Physeter macrocephalus (n = 7). Out of 118 strandings, 93 occurred in the south coast (78.8 %), 23 in the north coast (19.5%) and 2 (1.7%) in the north-west coast of the State. The highest frequency of strandings occurred during the months of August to March and the maximum number of strandings occurred from 2000 onwards, as a consequence of the intense monitoring of the Pequenos Cetáceos Project in Rio Grande do Norte

Potencial terapêutico de um heparinóide isolado do invertebrado marinho Litopenaeus vannamei

The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases

Efeito do consumo de farinha de linhaça (Linum usitatissimum) no crescimento de ratos Wistar e relação com a digestibilidade de globulinas e fatores antinutricionais protéicos

Linseed is an important oilseed consumed raw as nutritional supplement, that although represents a rich source of nutrients, its nutritional value could be impaired due to the presence of antinutritional factors. In this study, protein fractions from raw linseed flour were extracted and isolated being obtained 12% of albumins, 82% of globulins, 5% of glutelins and 1% of prolamins. These proteins were visualized by SDS-PAGE and albumins showed low molecular mass protein bands around 21 kDa and minor bands, similar to that of trypsin inhibitor; Globulins presented protein bands with high molecular masses, which possibly are constituents of multimeric proteins, such as legumins. After determination of the centesimal composition of raw linseed, it was used as exclusive protein source for young rats to evaluate its effect on animal growth. The results showed negative effects on rat growth (weight gain 73% less than the control group) and reduction of intestinal villus (35%), that could be related with in vitro and in vivo globulin digestibility and proteinaceous antinutritional factors (mammalian digestive enzymes inhibitors and lectins) in albumin fraction. Native globulins showed, by SDS-PAGE, low susceptibility in vitro to trypsin and chymotrypsin, however presented high degradation by pancreatin. Thermal treatment of globulins for 5 and 15 minutes at 100ºC improved considerably its digestibility by trypsin and pancreatin. Globulins presented 93.2% in vivo digestibility, similar to the control protein. Albumin fraction had high trypsin inhibition activity (100%) and chymotrypsin inhibition of 28.3%; haemagglutinating activity was not detected. The results of this study indicate the negative action of trypsin inhibitors on animal growth, but can not be discarded its combined action with other antinutritional factors, which could compromise the raw linseed utilization as an alternative food

Clonagem e expressão do gene que codifica o inibidor de quimotripsina de Erythrina velutina WILLD. - caracterização e avaliação de seu potencial farmacológico

Proteinases are enzymes distributed widely founded in several organisms and perform many different functions, from maintaining homeostasis to the worsening of some diseases such as cancer, autoimmune diseases and infections. The proteins responsible of controlling the action of these enzymes are the inhibitors, that are classified based on their target proteases and are founded since simple organisms, such as bacteria, to higher organisms, such as larger plants and mammals. Plant proteinase inhibitors act by reducing or inactivating the activity of target proteases, thus, these proteins have been studied as potential tools in the treatment of diseases related to protease activities. In this context, an inhibitor of chymotrypsin from Erythrina velutina, called EvCI was previously purified and it was observed that this protein plays in vitro anticoagulant activity and anti-inflammatory activity in in vivo model. Aiming to reduce the environmental impact caused by the purification EvCI in high amounts and to facilitate the process of obtaining this protein, the recombinant chymotrypsin inhibitor from Eryhrina velutina was produced after cloning and expression in Escherichia coli. The bacteria were grown in LB medium and after induction of the expression this material was subjected to procedures for cell lysis and the product was applied on Nickel-affinity column. The proteins adsorbed were digested by thrombin and applied on Chymotrypsin-Sepharose affinity column, obtaining the purified inhibitor, named recEvCI. After electrophoresis, the recombinant inhibitor showed an approximately molecular mass of 17 kDa, and reduced the chymotrypsin and elastase activities in vitro. The recombinant inhibitor was sequenced and was found similar amino acids residues when compared to other inhibitors deposited in the database, with some modifications. recEvCI showed high stability under pH variations and reducing conditions, maintaining its activity around 80%. This protein increased the blood coagulation time in vitro by acting on the intrinsic pathway and did not show cytotoxicity against strains of mouse 3T3 fibroblasts and RAW 264.7 macrophages. recEvCI showed microbicide activity related to release of nitric oxide and consequently the activation of macrophages, futhermore having proinflammatory effects assessed by increased release of TNF-α. These results indicate that recEvCI can be biotechnologically used as a new tool in the control of coagulation-related diseases as well as can be an activating agent of the immune system in immunosuppressed individuals

Purificação e caracterização de uma ß-N-acetillhexosaminadase extraída do mamífero marinho Sotalia fluviatilis

This report shows 2232 times purification of a βNAcetylhexosaminidase from hepatic extracts from the sea mammal Sotalia fluviatilis homogenate with final recovery of 8,4%. Sequenced steps were utilized for enzyme purification: ammonium sulfate fractionation, Biogel A 1.5 m, chitin, DEAESepharose and hydroxyapatite chromatographies. The protein molecular mass was estimated in 10 kDa using SDSPAGE and confirmed by MALDITOF. It was found to have an optimal pH of 5.0 and a temperature of 60°C. Using pnitrophenylNAcetylβDglycosaminide apparent Km and Vmax values were of 2.72 mM and 0.572 nmol/mg/min, respectively. The enzyme was inhibited by mercury chloride (HgCl2) and sodium dodecil sulfate (SDS)