Papel da localização da carga em determinação dos parâmetros do canal iônico

To aureus α-HL channel, we used the cysteine-scanning mutagenesis technique. Twenty-four mutants were produced from the substitution of a single aminoacid of the primary structure of the α-HL pro this yzed after the incorporation of a mutant channel in planar lipid bilayer membranes. The modified proteins were studied in the absence and presence of watersoluble specific sulphydryl-specific reagents, in order to introduce a strong positive or negative harge at positions of substitution. The introduction of a negative charge in the stem region onverted the selectivity of the channel from weak anionic to more cationic. However, the troduction of a positive charge increased its selectivity to the anion. The degree of these alterations was inversely dependent on the channel radius at the position of the introduced harge (selectivity). As to the asymmetry of the conductance-voltage, the influence of the harge was more complex. The introduction of the negative charge in the stem region (the trans art of the pore) provoked a decrease. The intensity of these alterations depended on the radius, and on the type of free charge at the pore entrance. These results suggest that the free charge at surrounds the pore wall is responsible for the cation-anion selectivity of the channel. The istribution of the charges between the entrances is crucial for determining the asymmetry of e conductance-voltage curves. We hope that these results serve as a model for studies with other nanometric channels, in biological or planar lipid bilayer membranes or in iotechnological applications

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

Produção e análise do desempenho óptico e elétrico de filmes finos de SnO2:F para aplicações fotovoltaicas

Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq

Análise do grau de proteção do Aquífero Barreiras em perímetros sob fertilização química a partir de dados hidrogeofísicos - área da bacia do Rio Catu-RN

The use of chemical fertilization in arable perimeters provides increased productivity, though it can eventually lead to a qualitative depreciation of groundwater sources, especially if such sources are unconfined in nature. In this context, this thesis presents results from an analysis of the level of natural protection of the Barreiras Aquifer in an area located on the eastern coast of the Rio Grande do Norte State - Brazil. Such an aquifer is clastic in nature and has an unconfined hydraulic character, which clearly makes it susceptible to contamination from surface ground loads with contaminants associated with the leaching of excess fertilizers not absorbed by ground vegetation. The methodology used was based on the use of hydro-geophysical data, particularly inverse models of vertical electrical soundings (VES) and information from well profiles, allowing the acquisition of longitudinal conductance cartographies (S), data in mili-Siemens (mS), and the vulnerability of the aquifer. Such maps were prepared with emphasis to the unsaturated overlying zone, highlighting in particular its thickness and occurrence of clay lithologies. Thus, the longitudinal conductance and aquifer vulnerability reveal areas more susceptible to contamination in the northeast and east-central sections of the study area, with values equal to or less than 10mS and greater than or equal to 0,50, respectively. On the other hand, the southwestern section proved to be less susceptible to contamination, whose longitudinal conductance and vulnerability indices are greater than or equal to 30mS and less than or equal to 0,40, respectively.

Análise do grau de proteção do Aquífero Barreiras em perímetros sob fertilização química a partir de dados hidrogeofísicos - área da bacia do Rio Catu-RN

The use of chemical fertilization in arable perimeters provides increased productivity, though it can eventually lead to a qualitative depreciation of groundwater sources, especially if such sources are unconfined in nature. In this context, this thesis presents results from an analysis of the level of natural protection of the Barreiras Aquifer in an area located on the eastern coast of the Rio Grande do Norte State - Brazil. Such an aquifer is clastic in nature and has an unconfined hydraulic character, which clearly makes it susceptible to contamination from surface ground loads with contaminants associated with the leaching of excess fertilizers not absorbed by ground vegetation. The methodology used was based on the use of hydro-geophysical data, particularly inverse models of vertical electrical soundings (VES) and information from well profiles, allowing the acquisition of longitudinal conductance cartographies (S), data in mili-Siemens (mS), and the vulnerability of the aquifer. Such maps were prepared with emphasis to the unsaturated overlying zone, highlighting in particular its thickness and occurrence of clay lithologies. Thus, the longitudinal conductance and aquifer vulnerability reveal areas more susceptible to contamination in the northeast and east-central sections of the study area, with values equal to or less than 10mS and greater than or equal to 0,50, respectively. On the other hand, the southwestern section proved to be less susceptible to contamination, whose longitudinal conductance and vulnerability indices are greater than or equal to 30mS and less than or equal to 0,40, respectively.