Indicadores de cálcio e de voltagem codificados geneticamente na detecção de potenciais de ação e inputs sinápticos em cultura de neurônios hipocampais

Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ±8.0, R-GECO1: -57 ±2.5; VSFP: -60 ±3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ±18.3; GECO1-R: 128 ±15.3; VSFP: 94 ±14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ± 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ±6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ±19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee

Análise da resposta th17 em líquen plano oral

Th17 cells have been strongly associated with the pathogenesis of several autoimmune and inflammatory diseases. IL-17 and IL-23 are important cytokines associated with this lineage. The aim of this study was to analyze, through immunohistochemical methods, the immunoexpression of IL-17 and IL-23 in the inflammatory infiltrate of oral lichen planus (OLP) lesion compared to that of inflammatory fibrous hyperplasia (IFH) and between clinical forms reticular and erosive of OLP. The sample included 41 cases of OLP, of which 23 were reticular and 18 erosive and 10 cases of IFH. The results were subjected to nonparametric statistical tests with a 5% significance level. In OLP lesions histomorphological analysis, the most common findings were: hyperparakeratinization, specimens with atrophic epithelium in erosive clinical form (p = 0.011), epithelial projections in most of reticular type of lesions, in addition Civatte bodies were identified in most samples of both clinical forms. For immunohistochemistry analysis, five fields with strong immunoreactivity for IL-17 and IL-23 were photomicrographed at 400x magnification, images were transferred to a computer where with ImageJ software®, lymphocytes that exhibited cytoplasmic immunostaining for these cytokines were counted. A mean was established after for each case. There was no statistically significant difference in the number of imunopositive lymphocytes for IL-17 and IL-23 among the group of OLP and IFH group, however a larger amount of lymphocytes imunopositive for IL-17 was found in the LPO group (p = 0.079) and significantly higher amounts of those lymphocytes were found in the erosive OLP when compared to the group of reticular OLP and IFH (p = 0.019). Furthermore, a marker epithelial immunopositivity for IL-17 was observed in OLP group. Although the results of this study do not permit the forceful assertion about the participation of Th17 lineage in OLP lesions, the findings of immunopositive lymphocytes counting for IL-17 and IL-23, which are potent proinflammatory cytokines, together with the the marked epithelial immunopositivity found for IL-17 in this study, suggest a possible role of this lineage in the pathogenesis of this disorder

Resposta celular Th1 /Th2 na doença periodontal experimental, em ratos: um estudo imuno-histoquímico

Host response plays a major role in the pathogenesis of periodontal disease. Mediators such as inflammatory cytokines which are secreted during the immune response to bacterial challenges have ambiguous functions that may or may not lead to protection of the attacked tissue. In this context, experimental evidence suggests that T-helper 1 (Th-1) and T-helper 2 (Th-2) mediated responses are potentially important during the disease process. The aims of this study therefore were to further clarify the role played by Th2 cells during different time points of the active phase of periodontal disease, as well as, to investigate whether there was any evidence of a Th1 response in the periodontal disease microenvironment. Experimental periodontitis was induced in 30 Wistar male rats by placing cotton ligatures around the mandibular first molars. The rats were then randomly divided into two groups. Group1 (G1=15) and Group 2 (G2=15). In G1 the ligatures were maintained for 2 days, whereas in G2 the ligatures were left for 15 days, a time point that corresponds to the advanced stage of periodontal disease The contra-lateral teeth served as controls (no ligatures). Immunohistochemical investigation for the presence in gingival tissue of Th2 specific transcription factor (GATA3) and the subunit of the IFN-γ receptor was carried out after the disease induction period. Light microscopy analysis revealed a decrease in the expression of GATA-3 as bone loss progressed. On the other hand, although IFN-γ R1 was detected at an early stage of the active phase of disease its expression remained unaltered during the remaining period of the study. These results indicate that the Th2 response have a protective role during the pathogenesis of periodontal disease and that the progression of the periodontal disease is related with the unbalance of the responses Th1/Th2

Avaliação da resposta comportamental, morfofisiológica e produção de leite de cabras puras e mestiças da raça Saanen no semi-árido do Rio Grande do Norte

Goat breeding in the state of Rio Grande do Norte, Brazil has promising economic possibilities, with the proper handling of the natural resources. The introduction of specialized animals has been one of the ways used to improve herd genetics and increase productivity. However, climate has been one of the regional factors that most interferes with the adaptation of the new genetic prevalence resulting from the introduction of exotic breeds, because in their country of origin, the air temperature during most of the year is lower than the animals body temperature. With this in mind, the aim of this study was to characterize behavioral, physiological and morphological profiles and milk production of female Saanen goats belonging to different genetic groups raised in the semi-arid region of Rio Grande do Norte in Northeast Brazil. The study was conducted in the city of Lages (5° 42 00 S and 36° 14 41 W). We used 25 lactating female Saanen goats, distributed into 3 genetic groups: 5 purebred animals, 11 three-quarter bred and 9 half-bred. Behavioral observations were made over three consecutive days in the months of August and September, between 09:00 and 11:30h, when the animals were grazing. Physiological and meteorological data were recorded in the last three days of June, July, August and September at 05:00h and at 16:00h. In the semi-intensive breeding system, the animals from different genetic groups were similar in both field behavior and physiological response patterns. Although the purebred goats had longer hair, they did not show symptoms of thermal discomfort. Their white hair helped to reflect the short wavelength rays and thus eliminate those at the longer wave lengths. We concluded that the animals raised in the semi-intensive milk production system in this study seem to have adapted to the climatic conditions of the semi-arid region of Rio Grande do Norte, Brazil

Resposta comportamental e hormonal de machos não reprodutores de sagüi, callithrix jacchus, a estímulos sensoriais de filhotes não aparentados

Parental care in mammals is influenced by somatosensory stimuli from infants, such as vocalization and sight and by changes in the hormone levels of caretakers. The aim of this study was to evaluate the behavioral and hormonal responses of twelve non reproductive adult male common marmosets (Callithrix jacchus) to infant cues, vocalization recordings, sight and physical contact with newborn. Six out of twelve males had previous experience in caretaking. In article 1, adult males were exposed to newborn vocalizations for 10 minutes. On control condition no sound was presented. In article 2, males were tested on two conditions: a) Control: an empty acrylic transparent box (test box) was placed in male s cage for 15 minutes, and b) Experimental: males were exposed to newborns into a closed text box for 15 minutes. The cage was kept closed to prevented from tactile, smell and acoustic stimulation by the infant on common marmoset males. In article 3, males were exposed to an open or closed text box, which allowed or not their access to and social interaction with the infants. After each observation sessions, blood samples were collected to evaluate the cortisol levels of males. In all studies, behavioral response of adult males was significantly modified by newborns sight, vocalization and physical contact. Males approached and spent more time near the sound source and showed an increase in locomotion during sound exposure. Furthermore, males approached, smelled and spent more time near the test box when the newborn was inside it. There was no difference in behavioral pattern between experienced and non-experienced males in articles 1 and 2. In article 3, behavioral pattern of males was influence by previous caretaking experience. Experienced males recovered quicker and carried the infants more than the inexperienced ones. However, inexperienced males showed a decrease in recovery latency and an increase in carrying time after successive exposure to infants. Cortisol levels changed after exposure to infant s vocalization, especially for experienced adult males. Male hormonal profile was not affected by the sight of infants neither by their previous experienced in caretaking. The occurrence of social interaction between the caretaker and infant did not modify the hormonal profile of common marmoset males; however, as much as experienced males carried the infants their cortisol levels decreased. Thus, members of a social group or potential caretakers common marmosets exposed to sensory cues from dependent infant such as vocalization, sight, smell and physical contact, changed their behavioral and hormonal responses that are physiological modulators of parental behavior in common marmoset

Resposta humoral de bovinos vacinados contra as toxinas botulínicas tipos C e D em diferentes faixas etárias

Foi verificada pelo teste de ELISA indireto a resposta humoral contra os toxoides botulínicos C e D em bovinos de diferentes idades. O estudo envolveu 90 animais, que foram divididos em três grupos (n = 30), de acordo com a sua faixa etária; inferior a 2 anos de idade (G1), entre 2 e 5 anos (G2) e superior a 5 anos (G3). Os grupos experimentais foram vacinados com duas doses de vacina antibotulínica bivalente (C e D) comercial, nos dias 0 e 42 após a primo-vacinação (booster). Na avaliação, quando realizada 30 dias após o booster, os animais do G3 apresentaram maior produção de anticorpos (p < 0,05) em relação aos demais grupos. Entre o G1 e G2 não houve diferença significativa na resposta humoral contra a toxina C, no entanto, contra a toxina D, os animais do G1 apresentaram maior produção de anticorpos. Todos os grupos produziram uma resposta significativa de anticorpos contra as toxinas botulínicas após a 2ª dose da vacina bivalente comercial, principalmente contra o tipo D.

Indicadores da resposta ao estresse agudo associados com a familiaridade ao ambiente e aos procedimentos de banho e tosa em cães (Canis familiaris) da raça poodle

The stress responses can be parameters used in order to identify the welfare of animals. Behavior parameters can also be regarded as means to identify their relation to stressing situations. In this study, adult male and female poodle dogs, accustomed (group 1) or not (group 2) to the environment of a bath and grooming salon were analyzed through the plasmatic concentration of cortisol, cell counting of the immune system (total leukocytes and percentage of neutrophils and lymphocytes) and through observation of changes in behavior before and after the bath and grooming service. After arriving at the kennel, the dogs were taken to the bath and grooming facility, where they had their blood samples taken and were observed for ten minutes. The subsequent procedures consisted of the bath and grooming services and the collection of another blood samples. The research results were analyzed through non-parametric statistic tests and p < 0.05. Accustomed and non-accustomed dogs presented different hormonal and behavior responses: accustomed dogs presented an increasing of the cortisol level at the second blood samples collection, thus indicating stress towards the aforementioned procedures and presenting behavior responses which can be described as moderated stress. Non-accustomed dogs presented the same cortisol levels in both first and second samples suggesting that they were already reacting to the new environment. Dogs also shown behavioral responses which can be described as acute stress. Both male and female dogs showed similar immunological changes as well as different endocrine and behavioral profiles. Concerning aggression, this study demonstrates that biting the owner can be a predicting behavior of aggression during the bath and grooming service, male dogs were more aggressive than the female, and that males were more reactive to unknown caretaker. The results of this research propose that the familiarization of the animal with the bath and grooming environment and caretaker is important to the welfare of poodle dogs, especially to that of male dogs

Modelo de programação matemática para controle do crescimento da vegetação sob redes de distribuição de energia elétrica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Resposta de cultivares de arroz de sequeiro ao preparo do solo e à irrigação por aspersão

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resposta de cultivares de trigo ao nitrogênio em relação às fontes e épocas de aplicação sob plantio direto e irrigação por aspersão

A adubação nitrogenada é de suma importância para a cultura do trigo, já que o nitrogênio constitui um dos nutrientes mais exigidos por essa cultura e o rendimento desta é função direta da quantidade de nutrientes acumulados pela planta. Foram testados os adubos nitrogenados: sulfonitrato de amônio com inibidor de nitrificação, sulfato de amônio e uréia, na dose de 70 kg de N ha-1; em duas épocas de aplicação, na linha de semeadura ou em cobertura, além da testemunha que não recebeu nitrogênio como tratamento, em quatro cultivares de trigo irrigado: Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) 21, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) 22, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) 42 e IAC 370. O experimento foi conduzido sob irrigação por aspersão em dois anos (2005 e 2006) em área experimental pertencente à Faculdade de Engenharia de Ilha Solteira UNESP/São Paulo - Brasil. As fontes de nitrogênio sulfonitrato de amônio com inibidor de nitrificação, sulfato de amônio e uréia não diferiram entre si, porém foram superiores à testemunha, em relação à produtividade de grãos. A aplicação do N todo em cobertura proporcionou aumento na produtividade de grãos. O comportamento das cultivares quanto aos componentes de produção e produtividade foram dependentes do ano em estudo.

Resposta de cultivares de arroz a doses de nitrogênio e do regulador de crescimento cloreto de clormequat

O objetivo deste trabalho foi avaliar os efeitos de doses de nitrogênio (0, 50, 100 e 150 kg ha-1) e do regulador de crescimento cloreto de clormequat (0, 1 e 2 L ha-1), em algumas características agronômicas de cultivares de arroz IAC 201 e IAC 202. O delineamento experimental foi o de blocos ao acaso, com quatro repetições. O N foi aplicado em cobertura, no estádio da diferenciação floral, e o regulador de crescimento foi aplicado parceladamente, aos 20 e 30 dias após a emergência das plântulas de arroz (perfilhamento). Na safra 2001/2002, houve efeito significativo de N para altura de plantas, comprimento da panícula, espiguetas por panícula, massa de 100 grãos e produtividade de grãos. Houve efeito do regulador de crescimento sobre o comprimento da panícula e espiguetas por panícula. A maior produtividade foi a da cultivar IAC 202. Na safra 2002/2003, houve efeito de N para altura de plantas, massa de 100 grãos, fertilidade das espiguetas e produtividade de grãos. O regulador de crescimento não exerceu efeito nas características testadas, e a cultivar IAC 202 foi novamente a mais produtiva.

Avaliação clínica da resposta do tratamento periodontal não-cirúrgico (Full mouth desinfection) de pacientes diabéticos e não diabéticos

Tem sido demonstrado que o diabetes influencia no desenvolvimento e progressão da doença periodontal. Acredita-se ainda que há uma relação bi-direcional entre o Diabetes Mellito e a Doença Periodontal. Por isso, o tratamento periodontal pode responder de forma diferente em pacientes com e sem o quadro de diabetes. O objetivo desse estudo foi avaliar clinicamente o efeito da terapia periodontal não cirúrgica em pacientes com periodontite e diabetes mellito (grupo teste) e sem o quadro de diabetes (grupo controle). Para isso, realizou-se o tratamento periodontal não-cirúrgico FMSRP (Full mouth Scalling and Root Planing) e verificou-se os parâmetro clínicos periodontais (profundidade de sondagem, nível clínico de inserção, mobilidade, índice gengival e índice de placa) no início do estudo e após 3 meses. Na análise estatística, o paciente foi considerado como unidade de análise (p<0,05). Para as variáveis categóricas utilizou-se o teste de Fisher. Nas comparações inter-grupo, foi utilizado o Mann-Whitney Test e para comparações intra-grupo (baseline e três meses) utilizou-se o Teste de Wilcoxon. Participaram 26 pacientes no grupo controle e 14 no grupo teste. O índice de placa era 71,20% no início do estudo para o grupo teste e ficou 47,12% no fim; já no controle, os valores eram de 48,52% passando para 37,50%. E o índice gengival no grupo teste no baseline era 42,67 diminuindo para 26,81 e no grupo controle diminuiu de 41,36 para 30,62. A profundidade de sondagem foi no grupo teste 2,71mm passando para 2,40mm; já no controle, os valores foram de 2,84mm diminuindo para 2,55mm. O grupo controle ganhou 0,34 mm de inserção e o grupo teste perdeu 0,44mm de inserção. A recessão gengival aumentou 0,33mm no grupo teste e 0,04mm no grupo controle após os 3 meses. Houve diferença significativa inter-grupo para o índice de placa e gengival no baseline, também foi encontrado na recessão após 3 meses; já intra-grupo verificou-se diferença significativa para todas as variáveis, exceto para o nível clinico de inserção e mobilidade. Quando se categorizou a profundidade de sondagem em ≤3mm, >3 e≤6mm e >6mm, não foi encontrado diferença entre os grupos, mas verificou-se diferença significativa entre os períodos tanto para o grupo teste como para o controle. A hemoglobina glicada no grupo controle foi de 5,90% e no teste aumentou de 7,79% para 8,10%. Portanto, verifica-se que há uma melhora dos parâmetros clínicos periodontais em ambos os grupos, contudo não se verificou uma diferença significativa entre eles. Não obstante, o FMSRP promove um efeito benéfico em relação à condição de saúde periodontal, melhora dos parâmetros clínicos periodontais, em curto prazo (3 meses) tanto em indivíduos diabéticos como em não diabéticos, não sendo possível observar um melhor quadro glicêmico nos diabéticos

Resposta da crotalária a épocas e subdoses de aplicação de glifosato

The use of glyphosate sub-lethal rates can be an alternative to reduce the plant height of sunn hemp, facilitating its mechanical harvest. However, little known on the other effects that its application can result in this culture. This work aimed to evaluate the influence of application times (10; 20 and 30 days after the emergence) and glyphosate sub-lethal rates (0; 30; 60; 90 and 120 g ha(-1)), on the vegetative and reproductive development of Crotalaria juncea cultivated in no-tillage system. The experiment was conducted between the january and june months of year 2010, in Selviria, Mato Grosso do Sul State, Brazil, on a clayey Rhodic Haplustox (20 degrees 20' S and 51 degrees 24' W). The experiment design was randomized blocks in factorial arrangement 3 x 5 (application times x glyphosate rates), with four replicates. Were measured agronomic characters and productivity and the results undergone to the test F, being the times averages compared by Tukey test (P<0.05) and of rates by regression analysis. The stem diameter, seeds number per pod and mass of one thousand seeds not were affected by glyphosate application. Despite the glyphosate have provided reduction in plant height and increase of the branches number in Crotalaria juncea, not recommended the sub-lethal rates application of product per of affect straightly the culture productivity.

O problema do caixeiro viajante alugador : um estudo algorítmico

The Car Rental Salesman Problem (CaRS) is a variant of the classical Traveling Salesman Problem which was not described in the literature where a tour of visits can be decomposed into contiguous paths that may be performed in different rental cars. The aim is to determine the Hamiltonian cycle that results in a final minimum cost, considering the cost of the route added to the cost of an expected penalty paid for each exchange of vehicles on the route. This penalty is due to the return of the car dropped to the base. This paper introduces the general problem and illustrates some examples, also featuring some of its associated variants. An overview of the complexity of this combinatorial problem is also outlined, to justify their classification in the NPhard class. A database of instances for the problem is presented, describing the methodology of its constitution. The presented problem is also the subject of a study based on experimental algorithmic implementation of six metaheuristic solutions, representing adaptations of the best of state-of-the-art heuristic programming. New neighborhoods, construction procedures, search operators, evolutionary agents, cooperation by multi-pheromone are created for this problem. Furtermore, computational experiments and comparative performance tests are conducted on a sample of 60 instances of the created database, aiming to offer a algorithm with an efficient solution for this problem. These results will illustrate the best performance reached by the transgenetic algorithm in all instances of the dataset

Resposta de Piaget a Vygotsky: convergências e divergências teóricas

Neste trabalho, ao analisar a resposta de Piaget às críticas de Vygotsky, procuramos salientar convergências e divergências teóricas básicas colocadas pelo primeiro em relação ao segundo. Para isso, num primeiro momento, analisamos os esclarecimentos de Piaget sobre a realidade do fenômeno psicológico do egocentrismo cognitivo e linguístico; num segundo momento, analisamos as relações entre as noções científicas e espontâneas na criança. Num terceiro momento, analisamos a posição diferenciada de Piaget sobre os mecanismos que explicam o desenvolvimento e progresso do conhecimento. Concluímos que, na resposta de Piaget a Vygotsky, existe clara diferenciação do seu sistema teórico.