Marta Pinto | Michigan State University (original) (raw)

Papers by Marta Pinto

Biological Chemistry, 2010

Mycobacterium tuberculosis infection continues to be a major cause of morbidity and mortality thr... more Mycobacterium tuberculosis infection continues to be a major cause of morbidity and mortality throughout the world. The vast complexity of the intracellular pathogen M. tuberculosis and the diverse mechanisms by which it can invade host cells highlight the importance of developing a fully protective vaccine. Our vaccine development strategy consists of including fragments from multiple mycobacterial proteins involved in cell invasion. The aim of this study was to identify high activity binding peptides (HABPs) in the immunogenic protein Rv1980c from M. tuberculosis H37Rv with the ability to inhibit mycobacterial invasion into U937 monocyte-derived macrophages and A549 cells. The presence and transcription of the Rv1980c gene was assessed in members belonging to the M. tuberculosis complex and other nontuberculous mycobacteria by PCR and RT-PCR, respectively. Cell surface localization was confirmed by immuno-electron microscopy. Three peptides binding with high activity to U937 cells and one to A549 cells were identified. HABPs 31100, 31101, and 31107 inhibited invasion of M. tuberculosis into A549 and U937 cells and therefore could be promising candidates for the design of a subunit-based antituberculous vaccine.

Journal of Computer-aided Molecular Design, 2004

Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell deat... more Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell death, is related to the generation and development of several types of cancer as well as to an elevated resistance to chemotherapeutic treatments. Given that synthetic peptide fragments of the BH3 domain are capable to bind to both proteins and induce apoptosis in cell-free systems and HeLa cells, small molecule non-peptide mimics of these peptides can be considered as a new therapeutic strategy for the treatment of diseases associated to a deficient apoptosis or resistant to the treatments with chemotherapeutic drugs. This strategy is supported by experimental evidences about the death of transformed cells and sensibilization of tumoral cells by the inhibition of the antiapoptotic proteins Bcl-2 and Bcl-xL. In the current work, these proteins complexed with X(16BH3), where X designates the proapoptotic proteins Bak, Bax, Bid and Hrk, have been modeled in order to establish a pharmacophoric hypothesis that must be present in any ligand capable of binding with the antiapoptotic proteins Bcl-2 and Bcl-xL. The pharmacophore is also used to explain the structural features of a set of new small molecule inhibitors of these antiapoptotic proteins.

Journal of Computer-aided Molecular Design, 2004

Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell deat... more Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell death, is related to the generation and development of several types of cancer as well as to an elevated resistance to chemotherapeutic treatments. Given that synthetic peptide fragments of the BH3 domain are capable to bind to both proteins and induce apoptosis in cell-free systems and HeLa cells, small molecule non-peptide mimics of these peptides can be considered as a new therapeutic strategy for the treatment of diseases associated to a deficient apoptosis or resistant to the treatments with chemotherapeutic drugs. This strategy is supported by experimental evidences about the death of transformed cells and sensibilization of tumoral cells by the inhibition of the antiapoptotic proteins Bcl-2 and Bcl-xL. In the current work, these proteins complexed with X(16BH3), where X designates the proapoptotic proteins Bak, Bax, Bid and Hrk, have been modeled in order to establish a pharmacophoric hypothesis that must be present in any ligand capable of binding with the antiapoptotic proteins Bcl-2 and Bcl-xL. The pharmacophore is also used to explain the structural features of a set of new small molecule inhibitors of these antiapoptotic proteins.

PLOS One, 2009

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin... more The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis. CO5 (to G.V., J.B., A.P.) from MCYT (Spain), CTQ2004 08077 CO2 (to J.B.) and BIO2007 67904 (to A.P.) from MEC (Spain), IB05 136 (to J.B.) from CEC Principado de Asturias, 2005SGR00883 (to A.P.) from Generalitat de Catalunya, DR06/0009 (to N.B.C.) from red Heracles ISCIII (Spain). Funding from Fundação Ciência e Tecnologia (FCT) (Portugal) through POCTI and POCI programmes (Programa Operacional Ciência e Inovação 2010) (to M.J.S.) and FEDER (Project POCI/SAU-MMO/57321/ 2004 to M.R.A.). POCTI/SAU NEU/58735/2004 (to A.M.D.), PTDC/SAU NEU/69123/2006 (to A.M.D.) and FEDER funds (to A.M.D.) are acknowledged. Diffraction data were collected at ESRF beam line ID14 1.

PLOS One, 2009

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin... more The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis. CO5 (to G.V., J.B., A.P.) from MCYT (Spain), CTQ2004 08077 CO2 (to J.B.) and BIO2007 67904 (to A.P.) from MEC (Spain), IB05 136 (to J.B.) from CEC Principado de Asturias, 2005SGR00883 (to A.P.) from Generalitat de Catalunya, DR06/0009 (to N.B.C.) from red Heracles ISCIII (Spain). Funding from Fundação Ciência e Tecnologia (FCT) (Portugal) through POCTI and POCI programmes (Programa Operacional Ciência e Inovação 2010) (to M.J.S.) and FEDER (Project POCI/SAU-MMO/57321/ 2004 to M.R.A.). POCTI/SAU NEU/58735/2004 (to A.M.D.), PTDC/SAU NEU/69123/2006 (to A.M.D.) and FEDER funds (to A.M.D.) are acknowledged. Diffraction data were collected at ESRF beam line ID14 1.

Neuroscience, 2009

Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating system... more Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating systems that include the caudal ventrolateral medulla (CVLM). The neuropeptide angiotensin II (Ang II) has multiple effects in the CNS and at the medulla oblongata. Here we evaluated the expression of angiotensin type 1 (AT(1)) receptors in spinally-projecting CVLM neurons, and tested the effect of direct application of exogenous Ang II in the CVLM on nociceptive behaviors. Although AT(1)-immunoreactive neurons occurred in the CVLM, only 3% of AT(1)-positive neurons were found to project to the dorsal horn, using double-immunodetection of the retrograde tracer cholera toxin subunit B. In behavioral studies, administration of Ang II (100 pmol) in the CVLM gave rise to hyperalgesia in both the tail-flick and formalin tests. This hyperalgesia was significantly attenuated by local administration of the AT(1) antagonist losartan. The present study demonstrates that Ang II can act on AT(1) receptors in the CVLM to modulate nociception. The effect on spinal nociceptive processing is likely indirect, since few AT(1)-expressing CVLM neurons were found to project to the spinal cord. The renin-angiotensin system may also play a role in other supraspinal areas implicated in pain modulation.

European Journal of Neuroscience, 2003

Modulation of nociceptive transmission at the brainstem involves several neurochemical systems. T... more Modulation of nociceptive transmission at the brainstem involves several neurochemical systems. The precise location and speci®c characteristics of nociceptive neurons activated in each system was never reported. In this study, the presence of GABA B , m-opioid, and neurokinin-1 (NK-1) receptors in brainstem nociceptive neurons was investigated by double-immunocytochemical detection of each receptor and noxious-evoked induction of the c-fos proto-oncogene. Noxious cutaneous mechanical stimulation signi®cantly increased the proportions of neurons double-labelled for Fos and GABA B receptors in several brainstem regions, namely, the reticular formation of the caudal ventrolateral medulla (VLMlat and VLMrf), lateral reticular nucleus, spinal trigeminal nucleus, pars caudalis (Sp 5 C), nucleus of the solitary tract, dorsal reticular nucleus, ventral reticular nucleus, raphe obscurus nucleus and dorsal parabrachial nucleus (DPB). For m-opioid receptors, the proportions of double-labelled neurons in noxious-stimulated animals were higher than in controls only in the VLMlat, VLMrf, Sp 5 C, DPB and A 5 noradrenergic cell group. As for the NK-1 receptor, no signi®cant differences were found between control and stimulated animals. According to these results, neurons expressing GABA B , m-opioid and NK-1 receptors at several pain control centres of the brainstem are differentially involved in processing nociceptive mechanical input. The data provide the de®nition of new supraspinal targets for selective modulation of nociceptive neurons in order to de®ne better strategies of pain control.

Molecular Therapy, 2005

Subcutaneous application of Herpes Simplex Virus type 1 (HSV-1) vectors mediating enkephalin tran... more Subcutaneous application of Herpes Simplex Virus type 1 (HSV-1) vectors mediating enkephalin transfer to the sensory neurons of the dorsal root ganglia, were shown to induce analgesia and reduce hyperalgesic reactions. These results prompted to use these vectors in the ...

Neuroscience, 2006

Secondary hyperalgesia in the monoarthritic rat is accompanied by a decrease in nociceptive activ... more Secondary hyperalgesia in the monoarthritic rat is accompanied by a decrease in nociceptive activation of spinal neurons expressing GABA(B) receptors and by the opposite effect in the cells expressing neurokinin 1 (NK1)-receptors. In order to ascertain the relative role of each receptor, the effects of intrathecal administration of SP-saporin (SP-SAP), baclofen or both were evaluated, using a model of secondary hyperalgesia that consists of mechanical stimulation of the hindlimb skin close to an inflamed joint. Four days after the induction of monoarthritis by intraarticular injection of Complete Freund's Adjuvant (CFA), a cannula was implanted at T(13)-L(1) level and 10 microl of saline or SP-SAP (10(-6) M) were intrathecally (i.t.) injected. Fourteen days after CFA-injection, half of the animals from each group received i.t. injections of 10 microl saline and the remainder were injected with the same volume of baclofen (1 microg). Ten minutes later, the animals were behaviorally evaluated by the von Frey test or submitted to noxious mechanical stimulation to analyze c-fos expression. The von Frey thresholds increased after the treatments, but more pronouncedly after baclofen or SP-SAP plus baclofen. In segments L(2)-L(3), the spinal area that receives input from the stimulated skin close to the inflamed joint, the numbers of Fos-immunoreactive neurons were reduced after the three treatments both in the superficial and deep dorsal horn. In segments T(13)-L(1), the numbers of Fos-immunoreactive neurons were significantly reduced after treatment with SP-SAP plus baclofen in both dorsal horn regions, and in the deep dorsal horn after baclofen treatment. We conclude that both GABA(B) and NK1 receptors of spinal dorsal horn neurons participate in secondary hyperalgesia in the monoarthritic rat, although the decrease in GABA inhibition appears to play a more important role than the increase in SP-mediated effects.

Journal of Comparative Neurology, 2008

Several brain areas modulate pain transmission through direct projections to the spinal cord. The... more Several brain areas modulate pain transmission through direct projections to the spinal cord. The descending modulation is exerted by neurotransmitters acting both at spinally projecting neurons and at interneurons that target the projection neurons. We analyzed the expression of μ-opioid (MOR), γ-aminobutyric acid GABAB, and NK1 receptors in spinally projecting neurons of major medullary pain control areas of the rat: rostroventromedial medulla (RVM), dorsal reticular nucleus (DRt), nucleus of the solitary tract, ventral reticular nucleus, and lateralmost part of the caudal ventrolateral medulla. The retrograde tracer cholera toxin subunit B (CTb) was injected into the spinal dorsal horn, and medullary sections were processed by double immunocytochemistry for CTb and each receptor. The RVM contained the majority of double-labeled neurons followed by the DRt. In general, high percentages of MOR- and NK1-expressing neurons were retrogradely labeled, whereas GABAB receptors were mainly expressed in neurons that were not labeled from the cord. The results suggest that MOR and NK1 receptors play an important role in direct and indirect control of descending modulation. The co-localization of MOR and GABAB in DRt neurons also demonstrated by the present study suggests that the pronociceptive effects of this nucleus may be controlled by local opoidergic and GABAergic inhibition of the pronociception increased during chronic pain. J. Comp. Neurol. 510:175–187, 2008. © 2008 Wiley-Liss, Inc.

Molecular and Cellular Neuroscience, 2008

During chronic pain, the supraspinal pain modulatory system undergoes plastic changes with enhanc... more During chronic pain, the supraspinal pain modulatory system undergoes plastic changes with enhancement of facilitation transmission at the spinal cord. The changes induced by chronic pain at descending modulation often affect opioidergic modulation, and were never described for a key facilitatory component of the system, the dorsal reticular nucleus (DRt). Neurochemical characterization of the DRt-spinal pathway showed that δ-opioid receptors are positioned as to indirectly modulate the activity of non-projecting DRt neurons, whereas neurons expressing μ-opioid receptors project to the spinal dorsal horn or act as interneurons, the latter of which co-expressing GABA B receptors. In monoarthritic rats, the expression of μopioid receptors decreased in the DRt whereas the levels of endogenous enkephalin remained unaltered. To increase the opioidergic inhibition of the DRt, we locally injected selective agonists of δand μ-opioid receptors or a viral vector containing the human preproenkephalin transgene. Injection of the Herpes Simplex viral vector encoding preproenkephalin induced thermal hypoalgesia in non-inflamed animals and hyperalgesia in monoarthritic rats. The opioid agonists [D-Ala 2 , Glu 4 ]-deltorphin (DELT) and [D-Ala 2 , NMePhe 4 Gly-ol 5 ]-enkephalin (DAMGO) induced thermal hyperalgesia in both non-inflamed and monoarthritic rats, but with lower doses in the latter group. The present study shows that opioidergic neurons at the DRt are modulated by GABAergic cells herein controlling the descending facilitation of pain transmission. The DRt exhibits plastic changes during chronic inflammatory pain, with decrease opioid receptor expression which may account for increased descending facilitation during chronic pain.

Neuroscience, 2007

Chronic inflammatory pain induces short- and long-term central changes, which have been mainly st... more Chronic inflammatory pain induces short- and long-term central changes, which have been mainly studied at the spinal cord level. Supraspinal pain control centers intrinsically connected with the dorsal horn are also prone to be affected by chronic inflammatory pain. C-fos expression was used as a neuronal activation marker at spinal and supraspinal levels to i) compare acute and chronic articular inflammation, and ii) analyze the effects of brief innocuous or noxious stimulation of a chronically inflamed joint. Acute articular inflammation was induced by an inflammatory soup with prostaglandin E(2) and bradykinin, both at 10(-5) M. Chronic articular inflammation consisted of 14 days of monoarthritis. Early c-fos expression was studied 4 min after inflammatory soup injection or stimulation of the arthritic joint whereas late c-fos expression was evaluated 2 h after those stimuli. At the spinal cord, the analysis was focused on the dorsal horn (laminae I-V) and supraspinally, five major regions of the endogenous pain control system were considered: the caudal ventrolateral medulla (VLM), the dorsal reticular nucleus (DRt), the ventral reticular nucleus (VRt), the nucleus of the solitary tract (Sol) and the rostroventromedial medulla (RVM). Acute articular inflammation induced early and late increases in c-fos expression at the spinal level and late increases supraspinally whereas the effects of monoarthritis were more moderate and restricted to the spinal cord. When monoarthritic animals were subjected to gentle touch or bending of the joint, early increases in c-fos expression were detected supraspinally, but not at the spinal level. In this region, noxious mechanical stimulation induced late increases in non-inflamed animals and both early and late increases in monoarthritic rats. Supraspinally, noxious stimulation induced only late increases in c-fos expression. The present results show complex differences in the patterns of c-fos expression between the spinal cord and medullary areas of the pain control system during articular inflammation, which indicate that the somatosensory system is differentially affected by the installation of chronic pain.

Neuroscience Letters, 2006

Chronic pain induces functional alterations of the endogenous pain control system namely in the m... more Chronic pain induces functional alterations of the endogenous pain control system namely in the modulation of nociceptive transmission at the spinal cord. We used the c-fos expression as a tool to study correlated neuronal activation, induced by bending the inflamed paw of monoarthritic animals, between the spinal dorsal horn and medullary centers belonging to the endogenous pain control system, namely the lateralmost reticular formation of the ventrolateral medulla (VLMlat), the lateral reticular nucleus (LRt), the dorsal reticular nucleus (DRt), the nucleus tractus solitarius (Sol) and the rostroventromedial medulla (RVM). Awake monoarthritic rats were subjected to 4 min of paw bending followed by anaesthesia and perfusion either immediately or 2 h later. The numbers of Fos immunoreactive neurons in the spinal dorsal horn and in the medulla oblongata were significantly correlated mainly immediately after stimulation: lamina I correlated with the VLMlat, LRt, Sol and RVM; lamina II correlated with the VLMlat, LRt and Sol; and laminae IV-V correlated with the VLMlat and LRt. Between medullary pain control centers significant correlations occurred immediately and 2 h after bending at the VLMlat-Sol and LRt-Sol, at the VLMlat-LRt and VLMlat-RVM in animals perfused immediately, and at the VLMlat-DRt and LRt-RVM in animals perfused 2 h later. These data demonstrate that the mobilization of a chronically inflamed paw triggers intense correlated neuronal activity in several areas of the somatosensory system, indicating functional relevant links in pain control.

Neuroscience, 2005

The neurochemical changes that operate in nociceptive spinal cord circuits during secondary hyper... more The neurochemical changes that operate in nociceptive spinal cord circuits during secondary hyperalgesia are largely unknown, in particular with respect to the balance between excitatory and inhibitory neurotransmission. In this study we evaluated the expression of NK1 and GABA B receptors in nociceptive spinal neurons in a model of secondary hyperalgesia consisting of noxious mechanical stimulation of the hindlimb skin close to a joint chronically inflamed by complete Freund's adjuvant. In spinal segments receiving input from that skin area, Fos-immunodetection was combined with immunocytochemistry for NK1 receptors, GABA B receptors or both receptors. In control and monoarthritic animals, neurons double-labeled for Fos and each receptor occurred mainly in laminae I and IV-V. In lamina I, the percentage of NK1 neurons expressing Fos was higher in monoarthritics while lower percentages of GABA B neurons expressed Fos. The percentage of Fos-positive cells expressing NK1 immunoreaction did not change in monoarthritics but that of Fos cells with GABA B immunoreaction was lower in these animals. In laminae IV-V, a large increase in Fos expression was detected in monoarthritic rats but the relative proportions of Fos-positive neurons expressing each receptor were similar in the two groups. Co-localization of NK1 and GABA B receptors occurred only in lamina I neurons in both experimental groups with no differences between control and monoarthritic animals in the percentages of Fos-positive neurons that expressed the receptors. Considering the participation of lamina I neurons bearing NK1 and GABA B receptors in several spinofugal systems, it is possible that the imbalance between excitatory and inhibitory actions exerted, respectively, by substance P and GABA may subserve secondary hyperalgesia by increasing ascending transmission of nociceptive input. (I. Tavares). Abbreviations: CFA, complete Freund's adjuvant; DF, dorsal funiculus; IR, immunoreactive; PBS, phosphate-buffered saline; PBST, phosphatebuffered saline with 0.3% Triton X-100; SP, substance P; WDR, widedynamic range neurons. Neuroscience 132 (2005) 905-916 0306-4522/05$30.00ϩ0.00

European Journal of Neuroscience, 2008

Herpes Simplex Virus type 1 (HSV-1) vectors are known to inhibit nociceptive transmission at the ... more Herpes Simplex Virus type 1 (HSV-1) vectors are known to inhibit nociceptive transmission at the spinal cord after peripheral applications. Similar approaches may also be useful when applied at the supraspinal pain control system as the system includes pronociceptive (facilitatory) components. We performed a study aimed to analyse the migration of HSV-1 along with the inhibition of pronociception from the medullary dorsal reticular nucleus (DRt), a major facilitatory component of the supraspinal pain control system. To study the migration, a HSV-1 vector expressing lacZ under control of the human cytomegalovirus (hCMV) promoter was injected in the DRt and the expression of β-galactosidase (β-gal) was detected at 2, 4, 7, 10 and 14 days. Numerous β-gal-immunoreactive neurons were observed at the injection site until day 4, and at some of the brain areas projecting to the DRt until day 7. To block the pronociceptive effects of the DRt, a HSV-1 vector expressing the preproenkephalin transgene, under the control of hCMV promoter, was injected into the DRt. Behavioural evaluation was performed at the time-points referred above, using the paw withdrawal latency test to evaluate thermal nociceptive responses. Anti-hyperalgesic effects persisted during 4 days, decreasing after that time-point. The present study demonstrates that selective migration of HSV-1 should be considered in gene therapy strategies based on HSV-1 injections into the brain. The study also shows that it is possible to decrease pain facilitation from the brain using opioidergic inhibition of pronociceptive supraspinal areas.

Brain Research, 2004

The nociceptive nature of spinal dorsal horn neurons expressing NK1 and g-aminobutyric acid (GABA... more The nociceptive nature of spinal dorsal horn neurons expressing NK1 and g-aminobutyric acid (GABA) B receptors was evaluated in the rat. Immunodetection of the Fos protein, induced by noxious mechanical stimulation of the skin, was combined with immunocytochemistry for NK1 or GABA B receptors (double-immunostaining study) or both receptors (triple-immunostaining study). Neurons double-labeled for Fos and for each receptor largely prevailed in lamina I. The proportions of Fos-positive cells immunostained for NK1 or GABA B receptors were higher in lamina I than in the remaining spinal laminae. More Fos-positive cells were immunoreactive (IR) for GABA B receptors than for NK1 in all dorsal horn laminae. In the triple-immunostaing study, co-localization of NK1 and GABA B receptors occurred only in lamina I and was higher in neurons expressing Fos. As to the morphological lamina I cell class, NK1-positive cells belonged mainly to the fusiform type while similar proportions of fusiform, pyramidal and flattened NK1 neurons expressed GABA B receptors. No differences were found between those cell types as to the degree of nociceptive activation. The present results suggest that the co-localization of NK1 and GABA B receptors is a common feature of fusiform, pyramidal and flattened neurons in lamina I. Considering the participation of the three cell classes in various ascending systems, it is concluded that a simultaneous action of substance P (SP) and GABA may play an important role in the modulation of nociceptive input supraspinally transmitted from lamina I. D

This article aims at showing how mathematical knowledge is constructed and learned by students di... more This article aims at showing how mathematical knowledge is constructed and learned by students digital natives of the 5th and 6th grade of II cycle that have lagged in the learning process through the of GoAnimante. The GoAnimate is a software that allows the creation of comics dynamically. Teachers who interact with the digital natives using the tools of the internet get gains in learning and achieve improvement in the relationship between student, teacher and discipline. Based on a qualitative research, as proposed application, whether to identify the degree of efficiency of work to be performed with the group of students nominated by Jose Honorio Rodrigues College (procedure), using primarily a measure to transmit an initial assessment domain that participants have the concepts and notions of mathematics involved in the comics. After the procedure, there will be a further measure, which refers to the same evaluation (the initial measurement). For the initial measurement, shall be elected some test items Proof Brazil, together with the Journals of recovery indicated by the Department of Mathematics Education.

Biological Chemistry, 2010

Mycobacterium tuberculosis infection continues to be a major cause of morbidity and mortality thr... more Mycobacterium tuberculosis infection continues to be a major cause of morbidity and mortality throughout the world. The vast complexity of the intracellular pathogen M. tuberculosis and the diverse mechanisms by which it can invade host cells highlight the importance of developing a fully protective vaccine. Our vaccine development strategy consists of including fragments from multiple mycobacterial proteins involved in cell invasion. The aim of this study was to identify high activity binding peptides (HABPs) in the immunogenic protein Rv1980c from M. tuberculosis H37Rv with the ability to inhibit mycobacterial invasion into U937 monocyte-derived macrophages and A549 cells. The presence and transcription of the Rv1980c gene was assessed in members belonging to the M. tuberculosis complex and other nontuberculous mycobacteria by PCR and RT-PCR, respectively. Cell surface localization was confirmed by immuno-electron microscopy. Three peptides binding with high activity to U937 cells and one to A549 cells were identified. HABPs 31100, 31101, and 31107 inhibited invasion of M. tuberculosis into A549 and U937 cells and therefore could be promising candidates for the design of a subunit-based antituberculous vaccine.

Journal of Computer-aided Molecular Design, 2004

Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell deat... more Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell death, is related to the generation and development of several types of cancer as well as to an elevated resistance to chemotherapeutic treatments. Given that synthetic peptide fragments of the BH3 domain are capable to bind to both proteins and induce apoptosis in cell-free systems and HeLa cells, small molecule non-peptide mimics of these peptides can be considered as a new therapeutic strategy for the treatment of diseases associated to a deficient apoptosis or resistant to the treatments with chemotherapeutic drugs. This strategy is supported by experimental evidences about the death of transformed cells and sensibilization of tumoral cells by the inhibition of the antiapoptotic proteins Bcl-2 and Bcl-xL. In the current work, these proteins complexed with X(16BH3), where X designates the proapoptotic proteins Bak, Bax, Bid and Hrk, have been modeled in order to establish a pharmacophoric hypothesis that must be present in any ligand capable of binding with the antiapoptotic proteins Bcl-2 and Bcl-xL. The pharmacophore is also used to explain the structural features of a set of new small molecule inhibitors of these antiapoptotic proteins.

Journal of Computer-aided Molecular Design, 2004

Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell deat... more Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell death, is related to the generation and development of several types of cancer as well as to an elevated resistance to chemotherapeutic treatments. Given that synthetic peptide fragments of the BH3 domain are capable to bind to both proteins and induce apoptosis in cell-free systems and HeLa cells, small molecule non-peptide mimics of these peptides can be considered as a new therapeutic strategy for the treatment of diseases associated to a deficient apoptosis or resistant to the treatments with chemotherapeutic drugs. This strategy is supported by experimental evidences about the death of transformed cells and sensibilization of tumoral cells by the inhibition of the antiapoptotic proteins Bcl-2 and Bcl-xL. In the current work, these proteins complexed with X(16BH3), where X designates the proapoptotic proteins Bak, Bax, Bid and Hrk, have been modeled in order to establish a pharmacophoric hypothesis that must be present in any ligand capable of binding with the antiapoptotic proteins Bcl-2 and Bcl-xL. The pharmacophore is also used to explain the structural features of a set of new small molecule inhibitors of these antiapoptotic proteins.

PLOS One, 2009

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin... more The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis. CO5 (to G.V., J.B., A.P.) from MCYT (Spain), CTQ2004 08077 CO2 (to J.B.) and BIO2007 67904 (to A.P.) from MEC (Spain), IB05 136 (to J.B.) from CEC Principado de Asturias, 2005SGR00883 (to A.P.) from Generalitat de Catalunya, DR06/0009 (to N.B.C.) from red Heracles ISCIII (Spain). Funding from Fundação Ciência e Tecnologia (FCT) (Portugal) through POCTI and POCI programmes (Programa Operacional Ciência e Inovação 2010) (to M.J.S.) and FEDER (Project POCI/SAU-MMO/57321/ 2004 to M.R.A.). POCTI/SAU NEU/58735/2004 (to A.M.D.), PTDC/SAU NEU/69123/2006 (to A.M.D.) and FEDER funds (to A.M.D.) are acknowledged. Diffraction data were collected at ESRF beam line ID14 1.

PLOS One, 2009

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin... more The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis. CO5 (to G.V., J.B., A.P.) from MCYT (Spain), CTQ2004 08077 CO2 (to J.B.) and BIO2007 67904 (to A.P.) from MEC (Spain), IB05 136 (to J.B.) from CEC Principado de Asturias, 2005SGR00883 (to A.P.) from Generalitat de Catalunya, DR06/0009 (to N.B.C.) from red Heracles ISCIII (Spain). Funding from Fundação Ciência e Tecnologia (FCT) (Portugal) through POCTI and POCI programmes (Programa Operacional Ciência e Inovação 2010) (to M.J.S.) and FEDER (Project POCI/SAU-MMO/57321/ 2004 to M.R.A.). POCTI/SAU NEU/58735/2004 (to A.M.D.), PTDC/SAU NEU/69123/2006 (to A.M.D.) and FEDER funds (to A.M.D.) are acknowledged. Diffraction data were collected at ESRF beam line ID14 1.

Neuroscience, 2009

Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating system... more Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating systems that include the caudal ventrolateral medulla (CVLM). The neuropeptide angiotensin II (Ang II) has multiple effects in the CNS and at the medulla oblongata. Here we evaluated the expression of angiotensin type 1 (AT(1)) receptors in spinally-projecting CVLM neurons, and tested the effect of direct application of exogenous Ang II in the CVLM on nociceptive behaviors. Although AT(1)-immunoreactive neurons occurred in the CVLM, only 3% of AT(1)-positive neurons were found to project to the dorsal horn, using double-immunodetection of the retrograde tracer cholera toxin subunit B. In behavioral studies, administration of Ang II (100 pmol) in the CVLM gave rise to hyperalgesia in both the tail-flick and formalin tests. This hyperalgesia was significantly attenuated by local administration of the AT(1) antagonist losartan. The present study demonstrates that Ang II can act on AT(1) receptors in the CVLM to modulate nociception. The effect on spinal nociceptive processing is likely indirect, since few AT(1)-expressing CVLM neurons were found to project to the spinal cord. The renin-angiotensin system may also play a role in other supraspinal areas implicated in pain modulation.

European Journal of Neuroscience, 2003

Modulation of nociceptive transmission at the brainstem involves several neurochemical systems. T... more Modulation of nociceptive transmission at the brainstem involves several neurochemical systems. The precise location and speci®c characteristics of nociceptive neurons activated in each system was never reported. In this study, the presence of GABA B , m-opioid, and neurokinin-1 (NK-1) receptors in brainstem nociceptive neurons was investigated by double-immunocytochemical detection of each receptor and noxious-evoked induction of the c-fos proto-oncogene. Noxious cutaneous mechanical stimulation signi®cantly increased the proportions of neurons double-labelled for Fos and GABA B receptors in several brainstem regions, namely, the reticular formation of the caudal ventrolateral medulla (VLMlat and VLMrf), lateral reticular nucleus, spinal trigeminal nucleus, pars caudalis (Sp 5 C), nucleus of the solitary tract, dorsal reticular nucleus, ventral reticular nucleus, raphe obscurus nucleus and dorsal parabrachial nucleus (DPB). For m-opioid receptors, the proportions of double-labelled neurons in noxious-stimulated animals were higher than in controls only in the VLMlat, VLMrf, Sp 5 C, DPB and A 5 noradrenergic cell group. As for the NK-1 receptor, no signi®cant differences were found between control and stimulated animals. According to these results, neurons expressing GABA B , m-opioid and NK-1 receptors at several pain control centres of the brainstem are differentially involved in processing nociceptive mechanical input. The data provide the de®nition of new supraspinal targets for selective modulation of nociceptive neurons in order to de®ne better strategies of pain control.

Molecular Therapy, 2005

Subcutaneous application of Herpes Simplex Virus type 1 (HSV-1) vectors mediating enkephalin tran... more Subcutaneous application of Herpes Simplex Virus type 1 (HSV-1) vectors mediating enkephalin transfer to the sensory neurons of the dorsal root ganglia, were shown to induce analgesia and reduce hyperalgesic reactions. These results prompted to use these vectors in the ...

Neuroscience, 2006

Secondary hyperalgesia in the monoarthritic rat is accompanied by a decrease in nociceptive activ... more Secondary hyperalgesia in the monoarthritic rat is accompanied by a decrease in nociceptive activation of spinal neurons expressing GABA(B) receptors and by the opposite effect in the cells expressing neurokinin 1 (NK1)-receptors. In order to ascertain the relative role of each receptor, the effects of intrathecal administration of SP-saporin (SP-SAP), baclofen or both were evaluated, using a model of secondary hyperalgesia that consists of mechanical stimulation of the hindlimb skin close to an inflamed joint. Four days after the induction of monoarthritis by intraarticular injection of Complete Freund's Adjuvant (CFA), a cannula was implanted at T(13)-L(1) level and 10 microl of saline or SP-SAP (10(-6) M) were intrathecally (i.t.) injected. Fourteen days after CFA-injection, half of the animals from each group received i.t. injections of 10 microl saline and the remainder were injected with the same volume of baclofen (1 microg). Ten minutes later, the animals were behaviorally evaluated by the von Frey test or submitted to noxious mechanical stimulation to analyze c-fos expression. The von Frey thresholds increased after the treatments, but more pronouncedly after baclofen or SP-SAP plus baclofen. In segments L(2)-L(3), the spinal area that receives input from the stimulated skin close to the inflamed joint, the numbers of Fos-immunoreactive neurons were reduced after the three treatments both in the superficial and deep dorsal horn. In segments T(13)-L(1), the numbers of Fos-immunoreactive neurons were significantly reduced after treatment with SP-SAP plus baclofen in both dorsal horn regions, and in the deep dorsal horn after baclofen treatment. We conclude that both GABA(B) and NK1 receptors of spinal dorsal horn neurons participate in secondary hyperalgesia in the monoarthritic rat, although the decrease in GABA inhibition appears to play a more important role than the increase in SP-mediated effects.

Journal of Comparative Neurology, 2008

Several brain areas modulate pain transmission through direct projections to the spinal cord. The... more Several brain areas modulate pain transmission through direct projections to the spinal cord. The descending modulation is exerted by neurotransmitters acting both at spinally projecting neurons and at interneurons that target the projection neurons. We analyzed the expression of μ-opioid (MOR), γ-aminobutyric acid GABAB, and NK1 receptors in spinally projecting neurons of major medullary pain control areas of the rat: rostroventromedial medulla (RVM), dorsal reticular nucleus (DRt), nucleus of the solitary tract, ventral reticular nucleus, and lateralmost part of the caudal ventrolateral medulla. The retrograde tracer cholera toxin subunit B (CTb) was injected into the spinal dorsal horn, and medullary sections were processed by double immunocytochemistry for CTb and each receptor. The RVM contained the majority of double-labeled neurons followed by the DRt. In general, high percentages of MOR- and NK1-expressing neurons were retrogradely labeled, whereas GABAB receptors were mainly expressed in neurons that were not labeled from the cord. The results suggest that MOR and NK1 receptors play an important role in direct and indirect control of descending modulation. The co-localization of MOR and GABAB in DRt neurons also demonstrated by the present study suggests that the pronociceptive effects of this nucleus may be controlled by local opoidergic and GABAergic inhibition of the pronociception increased during chronic pain. J. Comp. Neurol. 510:175–187, 2008. © 2008 Wiley-Liss, Inc.

Molecular and Cellular Neuroscience, 2008

During chronic pain, the supraspinal pain modulatory system undergoes plastic changes with enhanc... more During chronic pain, the supraspinal pain modulatory system undergoes plastic changes with enhancement of facilitation transmission at the spinal cord. The changes induced by chronic pain at descending modulation often affect opioidergic modulation, and were never described for a key facilitatory component of the system, the dorsal reticular nucleus (DRt). Neurochemical characterization of the DRt-spinal pathway showed that δ-opioid receptors are positioned as to indirectly modulate the activity of non-projecting DRt neurons, whereas neurons expressing μ-opioid receptors project to the spinal dorsal horn or act as interneurons, the latter of which co-expressing GABA B receptors. In monoarthritic rats, the expression of μopioid receptors decreased in the DRt whereas the levels of endogenous enkephalin remained unaltered. To increase the opioidergic inhibition of the DRt, we locally injected selective agonists of δand μ-opioid receptors or a viral vector containing the human preproenkephalin transgene. Injection of the Herpes Simplex viral vector encoding preproenkephalin induced thermal hypoalgesia in non-inflamed animals and hyperalgesia in monoarthritic rats. The opioid agonists [D-Ala 2 , Glu 4 ]-deltorphin (DELT) and [D-Ala 2 , NMePhe 4 Gly-ol 5 ]-enkephalin (DAMGO) induced thermal hyperalgesia in both non-inflamed and monoarthritic rats, but with lower doses in the latter group. The present study shows that opioidergic neurons at the DRt are modulated by GABAergic cells herein controlling the descending facilitation of pain transmission. The DRt exhibits plastic changes during chronic inflammatory pain, with decrease opioid receptor expression which may account for increased descending facilitation during chronic pain.

Neuroscience, 2007

Chronic inflammatory pain induces short- and long-term central changes, which have been mainly st... more Chronic inflammatory pain induces short- and long-term central changes, which have been mainly studied at the spinal cord level. Supraspinal pain control centers intrinsically connected with the dorsal horn are also prone to be affected by chronic inflammatory pain. C-fos expression was used as a neuronal activation marker at spinal and supraspinal levels to i) compare acute and chronic articular inflammation, and ii) analyze the effects of brief innocuous or noxious stimulation of a chronically inflamed joint. Acute articular inflammation was induced by an inflammatory soup with prostaglandin E(2) and bradykinin, both at 10(-5) M. Chronic articular inflammation consisted of 14 days of monoarthritis. Early c-fos expression was studied 4 min after inflammatory soup injection or stimulation of the arthritic joint whereas late c-fos expression was evaluated 2 h after those stimuli. At the spinal cord, the analysis was focused on the dorsal horn (laminae I-V) and supraspinally, five major regions of the endogenous pain control system were considered: the caudal ventrolateral medulla (VLM), the dorsal reticular nucleus (DRt), the ventral reticular nucleus (VRt), the nucleus of the solitary tract (Sol) and the rostroventromedial medulla (RVM). Acute articular inflammation induced early and late increases in c-fos expression at the spinal level and late increases supraspinally whereas the effects of monoarthritis were more moderate and restricted to the spinal cord. When monoarthritic animals were subjected to gentle touch or bending of the joint, early increases in c-fos expression were detected supraspinally, but not at the spinal level. In this region, noxious mechanical stimulation induced late increases in non-inflamed animals and both early and late increases in monoarthritic rats. Supraspinally, noxious stimulation induced only late increases in c-fos expression. The present results show complex differences in the patterns of c-fos expression between the spinal cord and medullary areas of the pain control system during articular inflammation, which indicate that the somatosensory system is differentially affected by the installation of chronic pain.

Neuroscience Letters, 2006

Chronic pain induces functional alterations of the endogenous pain control system namely in the m... more Chronic pain induces functional alterations of the endogenous pain control system namely in the modulation of nociceptive transmission at the spinal cord. We used the c-fos expression as a tool to study correlated neuronal activation, induced by bending the inflamed paw of monoarthritic animals, between the spinal dorsal horn and medullary centers belonging to the endogenous pain control system, namely the lateralmost reticular formation of the ventrolateral medulla (VLMlat), the lateral reticular nucleus (LRt), the dorsal reticular nucleus (DRt), the nucleus tractus solitarius (Sol) and the rostroventromedial medulla (RVM). Awake monoarthritic rats were subjected to 4 min of paw bending followed by anaesthesia and perfusion either immediately or 2 h later. The numbers of Fos immunoreactive neurons in the spinal dorsal horn and in the medulla oblongata were significantly correlated mainly immediately after stimulation: lamina I correlated with the VLMlat, LRt, Sol and RVM; lamina II correlated with the VLMlat, LRt and Sol; and laminae IV-V correlated with the VLMlat and LRt. Between medullary pain control centers significant correlations occurred immediately and 2 h after bending at the VLMlat-Sol and LRt-Sol, at the VLMlat-LRt and VLMlat-RVM in animals perfused immediately, and at the VLMlat-DRt and LRt-RVM in animals perfused 2 h later. These data demonstrate that the mobilization of a chronically inflamed paw triggers intense correlated neuronal activity in several areas of the somatosensory system, indicating functional relevant links in pain control.

Neuroscience, 2005

The neurochemical changes that operate in nociceptive spinal cord circuits during secondary hyper... more The neurochemical changes that operate in nociceptive spinal cord circuits during secondary hyperalgesia are largely unknown, in particular with respect to the balance between excitatory and inhibitory neurotransmission. In this study we evaluated the expression of NK1 and GABA B receptors in nociceptive spinal neurons in a model of secondary hyperalgesia consisting of noxious mechanical stimulation of the hindlimb skin close to a joint chronically inflamed by complete Freund's adjuvant. In spinal segments receiving input from that skin area, Fos-immunodetection was combined with immunocytochemistry for NK1 receptors, GABA B receptors or both receptors. In control and monoarthritic animals, neurons double-labeled for Fos and each receptor occurred mainly in laminae I and IV-V. In lamina I, the percentage of NK1 neurons expressing Fos was higher in monoarthritics while lower percentages of GABA B neurons expressed Fos. The percentage of Fos-positive cells expressing NK1 immunoreaction did not change in monoarthritics but that of Fos cells with GABA B immunoreaction was lower in these animals. In laminae IV-V, a large increase in Fos expression was detected in monoarthritic rats but the relative proportions of Fos-positive neurons expressing each receptor were similar in the two groups. Co-localization of NK1 and GABA B receptors occurred only in lamina I neurons in both experimental groups with no differences between control and monoarthritic animals in the percentages of Fos-positive neurons that expressed the receptors. Considering the participation of lamina I neurons bearing NK1 and GABA B receptors in several spinofugal systems, it is possible that the imbalance between excitatory and inhibitory actions exerted, respectively, by substance P and GABA may subserve secondary hyperalgesia by increasing ascending transmission of nociceptive input. (I. Tavares). Abbreviations: CFA, complete Freund's adjuvant; DF, dorsal funiculus; IR, immunoreactive; PBS, phosphate-buffered saline; PBST, phosphatebuffered saline with 0.3% Triton X-100; SP, substance P; WDR, widedynamic range neurons. Neuroscience 132 (2005) 905-916 0306-4522/05$30.00ϩ0.00

European Journal of Neuroscience, 2008

Herpes Simplex Virus type 1 (HSV-1) vectors are known to inhibit nociceptive transmission at the ... more Herpes Simplex Virus type 1 (HSV-1) vectors are known to inhibit nociceptive transmission at the spinal cord after peripheral applications. Similar approaches may also be useful when applied at the supraspinal pain control system as the system includes pronociceptive (facilitatory) components. We performed a study aimed to analyse the migration of HSV-1 along with the inhibition of pronociception from the medullary dorsal reticular nucleus (DRt), a major facilitatory component of the supraspinal pain control system. To study the migration, a HSV-1 vector expressing lacZ under control of the human cytomegalovirus (hCMV) promoter was injected in the DRt and the expression of β-galactosidase (β-gal) was detected at 2, 4, 7, 10 and 14 days. Numerous β-gal-immunoreactive neurons were observed at the injection site until day 4, and at some of the brain areas projecting to the DRt until day 7. To block the pronociceptive effects of the DRt, a HSV-1 vector expressing the preproenkephalin transgene, under the control of hCMV promoter, was injected into the DRt. Behavioural evaluation was performed at the time-points referred above, using the paw withdrawal latency test to evaluate thermal nociceptive responses. Anti-hyperalgesic effects persisted during 4 days, decreasing after that time-point. The present study demonstrates that selective migration of HSV-1 should be considered in gene therapy strategies based on HSV-1 injections into the brain. The study also shows that it is possible to decrease pain facilitation from the brain using opioidergic inhibition of pronociceptive supraspinal areas.

Brain Research, 2004

The nociceptive nature of spinal dorsal horn neurons expressing NK1 and g-aminobutyric acid (GABA... more The nociceptive nature of spinal dorsal horn neurons expressing NK1 and g-aminobutyric acid (GABA) B receptors was evaluated in the rat. Immunodetection of the Fos protein, induced by noxious mechanical stimulation of the skin, was combined with immunocytochemistry for NK1 or GABA B receptors (double-immunostaining study) or both receptors (triple-immunostaining study). Neurons double-labeled for Fos and for each receptor largely prevailed in lamina I. The proportions of Fos-positive cells immunostained for NK1 or GABA B receptors were higher in lamina I than in the remaining spinal laminae. More Fos-positive cells were immunoreactive (IR) for GABA B receptors than for NK1 in all dorsal horn laminae. In the triple-immunostaing study, co-localization of NK1 and GABA B receptors occurred only in lamina I and was higher in neurons expressing Fos. As to the morphological lamina I cell class, NK1-positive cells belonged mainly to the fusiform type while similar proportions of fusiform, pyramidal and flattened NK1 neurons expressed GABA B receptors. No differences were found between those cell types as to the degree of nociceptive activation. The present results suggest that the co-localization of NK1 and GABA B receptors is a common feature of fusiform, pyramidal and flattened neurons in lamina I. Considering the participation of the three cell classes in various ascending systems, it is concluded that a simultaneous action of substance P (SP) and GABA may play an important role in the modulation of nociceptive input supraspinally transmitted from lamina I. D

This article aims at showing how mathematical knowledge is constructed and learned by students di... more This article aims at showing how mathematical knowledge is constructed and learned by students digital natives of the 5th and 6th grade of II cycle that have lagged in the learning process through the of GoAnimante. The GoAnimate is a software that allows the creation of comics dynamically. Teachers who interact with the digital natives using the tools of the internet get gains in learning and achieve improvement in the relationship between student, teacher and discipline. Based on a qualitative research, as proposed application, whether to identify the degree of efficiency of work to be performed with the group of students nominated by Jose Honorio Rodrigues College (procedure), using primarily a measure to transmit an initial assessment domain that participants have the concepts and notions of mathematics involved in the comics. After the procedure, there will be a further measure, which refers to the same evaluation (the initial measurement). For the initial measurement, shall be elected some test items Proof Brazil, together with the Journals of recovery indicated by the Department of Mathematics Education.