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Nos moluscos sem concha ou naqueles em que a mesma estÃÂ presente na forma vestigial, como cefalÃÂpedes e gastrÃÂpodes opistobrÃÂnquios, a falta dessa proteÃÂÃÂo externa conduziu ÃÂ evoluÃÂÃÂo de mecanismos de defesa dominados pela secreÃÂÃÂo ou incorporaÃÂÃÂo de substÃÂncias quÃÂmicas bioativas. ProteÃÂnas sintetizadas de novo sÃÂo bons exemplos dessas molÃÂculas, tendo sido encontradas nas tintas de vÃÂrias espÃÂcies de lesmas do mar, particularmente do gÃÂnero Aplysia. Muitas jÃÂ revelaram pertencer a famÃÂlias de proteÃÂnas com atividade oxidÃÂstica de L-aminoÃÂcidos (LAAOs), grupo do qual fazem parte tambÃÂm proteÃÂnas bioativas encontradas nos venenos de serpentes. A caracterizaÃÂÃÂo, portanto, das atividades biolÃÂgicas e do perfil bioquÃÂmico dessas proteÃÂnas ÃÂ muito ÃÂtil para que possamos entender como se dÃÂo os mecanismos defensivos desses animais e quÃÂo difundidos esses sÃÂo, alÃÂm de proporcionar subsÃÂdios para sua aplicaÃÂÃÂo biomedicinal. Esse estudo visou a elucidaÃÂÃÂo dos mecanismos de aÃÂÃÂo da dactilomelina-P, proteÃÂna antibacteriana purificada da tinta de Aplysia dactylomela, e a comparaÃÂÃÂo da composiÃÂÃÂo protÃÂica e das atividades antimicrobianas das tintas liberadas por dois gastrÃÂpodes, A. dactylomela e Bursatella leachii, e por um cefalÃÂpode, Octopus sp.. O estudo revelou que a dactilomelina-P ÃÂ uma L-aminoÃÂcido oxidase capaz de oxidar L-lisina e L-arginina, com maior afinidade por L- arginina, com Km de 0,22 +- 0,16 mM/L para a L-lys e de 0,015 +- 0,01 Mm/L para a L-arg. Demonstrou tambÃÂm que sua atividade antibacteriana ÃÂ medida por esta propriedade, tendo o perÃÂxido de hidrogÃÂnio, gerado na oxidaÃÂÃÂo enzimÃÂticca, grande aprtipaÃÂÃÂo na inibiÃÂÃÂo bacteriana. A proteÃÂna se comporta de maneira bacteriostÃÂtica quando em meio nÃÂo suplementado com L-lisina e L-angilina, sendo bactericida na presenÃÂa destes. AlÃÂm disso, a atividade antibacteriana mostrou ser dependente da concentraÃÂÃÂo de aminoÃÂcidos disponibilizados para oxidaÃÂÃÂo pela proteÃÂna. A atividade Staphylococcus aureus deve ocorrer por meio de mecanismos que nÃÂo induzam danoso na morfologia celular, uma vez que nÃÂo foi observada qualquer alteraÃÂÃÂo morfolÃÂgica por microscopia de forÃÂa atÃÂmica. JÃÂ a anÃÂlise das trÃÂs tintas pro PAGE-SDS revelou a presenÃÂa, em todas elas, de bandas protÃÂicas de cerca de 60 kDa, alÃÂm de bandas de peso moleculares mais baixos. Por Western blot pode-se observar a existÃÂncia de identidade imunolÃÂgica entre proteinas das trÃÂs tintas, visto que anticorpos policlonais produzidos contra a dactilomelina-P reconheceram proteÃÂna(s) de cerca de 60 kDa na tinta de B. leachii e de cerca de 30 kDa na tinta de Octopus sp. Dentre as trÃÂs tintas, somente a tinta de A. dactylomela apresentou atividades antibacterianas e antifÃÂngica / In shelled molluscs or those in which it is present as vestigial as cephalopods and gastropods opistobrÃÂnquios, the lack of external protection led to the evolution of defense mechanisms dominated by the secretion or incorporation of bioactive chemicals. Proteins synthesized again are good examples of these molecules, were found in paints of various species of sea slugs, particularly the genus Aplysia. Many have already revealed belong to protein families with oxidÃÂstica activity of L-amino acids (LAAOs), a group which comprises also bioactive proteins found in snake venoms. The characterization therefore biological activities and biochemical profile of these proteins is very useful for us to understand how to give the defense mechanisms of animals and how widespread they are, in addition to providing subsidies to its application biomedicinal. This study aimed to elucidate the mechanisms of action of P-dactilomelina, antibacterial protein purified from Aplysia ink dactylomela, and comparison of protein composition and antimicrobial activities of paint released by two gastropods, A. dactylomela and Bursatella leachii, and a cephalopod, Octopus sp .. The study revealed that dactilomelina-P is an L-amino acid oxidase capable of oxidizing L-lysine and L-arginine, with a higher affinity for L-arginine, with a Km of 0.22 + - 0.16 mM / L for L -lys and 0.015 + - 0.01 mM / L L-arg. It also demonstrated that its antibacterial activity is measured by this property, with the hydrogen peroxide generated in the oxidation enzimÃÂticca, great aprtipaÃÂÃÂo in bacterial inhibition. The protein behaves in a non-bacteriostatic when supplemented with L-lysine and L-angilina, being in the presence of bactericidal. In addition, the antibacterial activity was shown to be dependent on the concentration of amino acids available for oxidation by the protein. Staphylococcus aureus activity must occur through mechanisms that do not induce damage to cell morphology, since we did not observe any morphological change by atomic force microscopy. The analysis of the three pro paint SDS-PAGE revealed the presence in them all, protein bands of about 60 kDa, and bands of lower molecular weight. For Western blot can observe the existence of immunologic identity between proteins of the three dyes, since polyclonal antibodies produced against P-dactilomelina recognized protein (s) of about 60 kDa in ink B. leachii and approximately 30 kDa in ink Octopus sp. Among the three dyes, only the ink of A. dactylomela showed antibacterial and antifungal activities

abstract: A direct Magnetic Resonance (MR)-based neural activity mapping technique with high spatial and temporal resolution may accelerate studies of brain functional organization. The most widely used technique for brain functional imaging is functional Magnetic Resonance Image (fMRI). The spatial resolution of fMRI is high. However, fMRI signals are highly influenced by the vasculature in each voxel and can be affected by capillary orientation and vessel size. Functional MRI analysis may, therefore, produce misleading results when voxels are nearby large vessels. Another problem in fMRI is that hemodynamic responses are slower than the neuronal activity. Therefore, temporal resolution is limited in fMRI. Furthermore, the correlation between neural activity and the hemodynamic response is not fully understood. fMRI can only be considered an indirect method of functional brain imaging. Another MR-based method of functional brain mapping is neuronal current magnetic resonance imaging (ncMRI), which has been studied over several years. However, the amplitude of these neuronal current signals is an order of magnitude smaller than the physiological noise. Works on ncMRI include simulation, phantom experiments, and studies in tissue including isolated ganglia, optic nerves, and human brains. However, ncMRI development has been hampered due to the extremely small signal amplitude, as well as the presence of confounding signals from hemodynamic changes and other physiological noise. Magnetic Resonance Electrical Impedance Tomography (MREIT) methods could have the potential for the detection of neuronal activity. In this technique, small external currents are applied to a body during MR scans. This current flow produces a magnetic field as well as an electric field. The altered magnetic flux density along the main magnetic field direction caused by this current flow can be obtained from phase images. When there is neural activity, the conductivity of the neural cell membrane changes and the current paths around the neurons change consequently. Neural spiking activity during external current injection, therefore, causes differential phase accumulation in MR data. Statistical analysis methods can be used to identify neuronal-current-induced magnetic field changes. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2019

One of the goals of neuroscience is to classify all of the neurons in the brain. Neuronal types can be defined using a combination of morphology, electrophysiology, and gene expression profiles. Gene expression profiles allow differentiation between cells that share similar characteristics. Leveraging the advantage of Berghia stephanieae (Gastropoda; Nudibranchia), which has around 28,000 neurons, I constructed high-throughput single-neuron transcriptomes for its whole brain. I produced a single-cell dissociation protocol and a custom data analysis pipeline for data of this nature. Around 129,000 cells were collected from 18 rhinophore ganglia and 20 circumesophageal ring ganglia (brain), consisting of the cerebropleural, pedal, and buccal ganglia. Messenger RNA libraries were constructed using the 10X Genomics’ Chromium platform. After library preparation, around 1,000 cells were recovered and sequenced. The HTStream package was utilized to trim off unwanted sequences from the raw reads and remove PCR duplicates and other contamination, then the salmon alevin package was employed to construct gene-by-cell matrices containing all the transcripts for each gene in each cell. The Seurat pipeline was used to extract this expression data from the matrices, normalize it, and perform dimensionality reduction. The cells were clustered based on similarities in their gene expression profiles. The cells formed eight clusters on a UMAP graph, each having distinct marker genes. Additionally, one cluster was composed of almost exclusively cells from the rhinophore ganglia, accounting for 30% of all rhinophore ganglion cells in the sample. Cells from the rhinophore ganglia are as heteregenous as cells from the rest of the brain, with cells forming six clusters. Cell populations that express the same neurotransmitter were identified for a wide range of both small-molecule neurotransmitters and neuropeptides. In a separate project, the locomotion of Berghia was recorded over 9 days with 2 lighting regimes: LD first and DD first. The results suggest that locomotion of Berghia is governed by circadian clock and that Berghia is nocturnal. Hunger state likely plays a role in modulating this circadian rhythm.

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