Mechanosensitive Ion Channel TMEM63A Gangs Up with Local Macrophages to Modulate Chronic Post-amputation Pain / 神经科学通报·英文版

Post-amputation pain causes great suffering to amputees, but still no effective drugs are available due to its elusive mechanisms. Our previous clinical studies found that surgical removal or radiofrequency treatment of the neuroma at the axotomized nerve stump effectively relieves the phantom pain afflicting patients after amputation. This indicated an essential role of the residual nerve stump in the formation of chronic post-amputation pain (CPAP). However, the molecular mechanism by which the residual nerve stump or neuroma is involved and regulates CPAP is still a mystery. In this study, we found that nociceptors expressed the mechanosensitive ion channel TMEM63A and macrophages infiltrated into the dorsal root ganglion (DRG) neurons worked synergistically to promote CPAP. Histology and qRT-PCR showed that TMEM63A was mainly expressed in mechanical pain-producing non-peptidergic nociceptors in the DRG, and the expression of TMEM63A increased significantly both in the neuroma from amputated patients and the DRG in a mouse model of tibial nerve transfer (TNT). Behavioral tests showed that the mechanical, heat, and cold sensitivity were not affected in the Tmem63a-/- mice in the naïve state, suggesting the basal pain was not affected. In the inflammatory and post-amputation state, the mechanical allodynia but not the heat hyperalgesia or cold allodynia was significantly decreased in Tmem63a-/- mice. Further study showed that there was severe neuronal injury and macrophage infiltration in the DRG, tibial nerve, residual stump, and the neuroma-like structure of the TNT mouse model, Consistent with this, expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β all increased dramatically in the DRG. Interestingly, the deletion of Tmem63a significantly reduced the macrophage infiltration in the DRG but not in the tibial nerve stump. Furthermore, the ablation of macrophages significantly reduced both the expression of Tmem63a and the mechanical allodynia in the TNT mouse model, indicating an interaction between nociceptors and macrophages, and that these two factors gang up together to regulate the formation of CPAP. This provides a new insight into the mechanisms underlying CPAP and potential drug targets its treatment.

Role of the Voltage-Gated Proton Channel Hv1 in Nervous Systems / 神经科学通报·英文版

Hv1 is the only voltage-gated proton-selective channel in mammalian cells. It contains a conserved voltage-sensor domain, shared by a large class of voltage-gated ion channels, but lacks a pore domain. Its primary role is to extrude protons from the cytoplasm upon pH reduction and membrane depolarization. The best-known function of Hv1 is the regulation of cytosolic pH and the nicotinamide adenine dinucleotide phosphate oxidase-dependent production of reactive oxygen species. Accumulating evidence indicates that Hv1 is expressed in nervous systems, in addition to immune cells and others. Here, we summarize the molecular properties, distribution, and physiological functions of Hv1 in the peripheral and central nervous systems. We describe the recently discovered functions of Hv1 in various neurological diseases, including brain or spinal cord injury, ischemic stroke, demyelinating diseases, and pain. We also summarize the current advances in the discovery and application of Hv1-targeted small molecules in neurological diseases. Finally, we discuss the current limitations of our understanding of Hv1 and suggest future research directions.

Research Progress of the Correlation between Caveolin and Unexpected Sudden Cardiac Death / 法医学杂志

Due to the negative autopsy and without cardiac structural abnormalities, unexpected sudden cardiac death （USCD） is always a tough issue for forensic pathological expertise. USCD may be associated with parts of fatal arrhythmic diseases. These arrhythmic diseases may be caused by disorders of cardiac ion channels or channel-related proteins. Caveolin can combine with multiple myocardial ion channel proteins through its scaffolding regions and plays an important role in maintaining the depolarization and repolarization of cardiac action potential. When the structure and function of caveolin are affected by gene mutations or abnormal protein expression, the functions of the regulated ion channels are correspondingly impaired, which leads to the occurrence of multiple channelopathies, arrhythmia or even sudden cardiac death. It is important to study the effects of caveolin on the functions of ion channels for exploring the mechanisms of malignant arrhythmia and sudden cardiac death.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+ cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover, FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs =0.317, P=0.006), hemoglobin levels (rs =0.210, P=0.049), and percentage of peripheral blood blasts (rs =0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs =–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higher FAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed that FAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Indirect orthodontic bonding - a modified technique for improved efficiency and precision

INTRODUCTION: The indirect bonding technique optimizes fixed appliance installation at the orthodontic office, ensuring precise bracket positioning, among other advantages. In this laboratory clinical phase, material and methods employed in creating the transfer tray are decisive to accuracy. OBJECTIVE: This article describes a simple, efficient and reproducible indirect bonding technique that allows the procedure to be carried out successfully. Variables influencing the orthodontic bonding are analyzed and discussed in order to aid professionals wishing to adopt the indirect bonding technique routinely in their clinical practice. .

INTRODUÇÃO: a técnica de colagem indireta prioriza a otimização do procedimento de montagem do aparelho fixo na clínica ortodôntica, assegurando, entre outras, vantagens relacionadas à precisão no posicionamento dos braquetes. Nesse procedimento clínico laboratorial, o material e o método de confecção da moldeira de transferência são determinantes no quesito precisão. OBJETIVO: este artigo descreve uma técnica de colagem indireta simples, eficiente e reprodutível, para que o procedimento possa ser realizado com sucesso. Variáveis que exercem influência sobre o procedimento são analisadas e discutidas, a fim de auxiliar o profissional a adotar, de forma rotineira, a técnica de colagem indireta em sua prática clínica. .

Uncoupling protein-2 mRNA expression in mice subjected to intermittent hypoxia / Expressão do mRNA da uncoupling protein-2 em camundongos submetidos à hipóxia intermitente

Objective: To investigate the effect of intermittent hypoxia-a model of obstructive sleep apnea (OSA)-on pancreatic expression of uncoupling protein-2 (UCP2), as well as on glycemic and lipid profiles, in C57BL mice. Methods: For 8 h/day over a 35-day period, male C57BL mice were exposed to intermittent hypoxia (hypoxia group) or to a sham procedure (normoxia group). The intermittent hypoxia condition involved exposing mice to an atmosphere of 92% N and 8% CO2 for 30 s, progressively reducing the fraction of inspired oxygen to 8 ± 1%, after which they were exposed to room air for 30 s and the cycle was repeated (480 cycles over the 8-h experimental period). Pancreases were dissected to isolate the islets. Real-time PCR was performed with TaqMan assays. Results: Expression of UCP2 mRNA in pancreatic islets was 20% higher in the normoxia group than in the hypoxia group (p = 0.11). Fasting serum insulin was higher in the hypoxia group than in the normoxia group (p = 0.01). The homeostasis model assessment of insulin resistance indicated that, in comparison with the control mice, the mice exposed to intermittent hypoxia showed 15% lower insulin resistance (p = 0.09) and 21% higher pancreatic β-cell function (p = 0.01). Immunohistochemical staining of the islets showed no significant differences between the two groups in terms of the area or intensity of α- and β-cell staining for insulin and glucagon. Conclusions: To our knowledge, this is the first report of the effect of intermittent hypoxia on UCP2 expression. Our findings suggest that UCP2 regulates insulin production in OSA. Further study of the role that UCP2 plays in the glycemic control of OSA patients is warranted. .

Objetivo: Investigar o efeito da hipóxia intermitente com um modelo de apneia obstrutiva do sono (AOS) sobre a expressão de uncoupling protein-2 (UCP2), assim como sobre perfis glicêmicos e lipídicos, em camundongos C57BL. Métodos: Camundongos C57BL machos foram expostos a hipóxia intermitente ou hipóxia simulada (grupo controle) 8 h/dia durante 35 dias. A condição de hipóxia intermitente envolveu a exposição dos camundongos a uma atmosfera de 92% de N e 8% de CO2 por 30 s, com redução progressiva de fração de O2 inspirado até 8 ± 1%, seguida por exposição a ar ambiente por 30 s e repetições do ciclo (480 ciclos no período experimental de 8 h). Os pâncreas foram dissecados para isolar as ilhotas. Foi realizada PCR em tempo real utilizando o método TaqMan. Resultados: A expressão do mRNA da UCP2 nas ilhotas pancreáticas foi 20% maior no grupo controle que no grupo hipóxia (p = 0,11). A insulina sérica de jejum foi maior no grupo hipóxia do que no grupo controle (p = 0,01). O modelo de avaliação da homeostase de resistência à insulina indicou que, em comparação com os camundongos controle, aqueles expostos à hipóxia intermitente apresentaram 15% menor resistência à insulina (p = 0,09) e 21% maior função das células beta (p = 0,01). A coloração das ilhotas pancreáticas por imuno-histoquímica não mostrou diferenças significativas entre os grupos em termos da área ou da intensidade das células alfa e beta, marcadas por insulina e glucagon. Conclusões: Segundo nosso conhecimento, esta é a primeira descrição do efeito da hipóxia intermitente sobre a expressão da UCP2. Nossos achados sugerem que UCP2 regula a produção de insulina na AOS. Futuras investigações sobre o papel da UCP2 no controle glicêmico em pacientes com AOS são justificadas. .

Brown adipose tissue: what have we learned since its recent identification in human adults / Tecido adiposo marrom: o que aprendemos desde sua recente identificação em humanos adultos

Brown adipose tissue, an essential organ for thermoregulation in small and hibernating mammals due to its mitochondrial uncoupling capacity, was until recently considered to be present in humans only in newborns. The identification of brown adipose tissue in adult humans since the development and use of positron emission tomography marked with 18-fluorodeoxyglucose (PET-FDG) has raised a series of doubts and questions about its real importance in our metabolism. In this review, we will discuss what we have learnt since its identification in humans as well as both new and old concepts, some of which have been marginalized for decades, such as diet-induced thermogenesis. Arq Bras Endocrinol Metab. 2014;58(9):889-99.

O tecido adiposo marrom, órgão essencial para a termorregulação de animais hibernantes e pequenos devido à sua capacidade desacopladora, era até poucos anos considerado presente apenas em recém-nascidos na espécie humana. A identificação do tecido adiposo marrom em adultos com o desenvolvimento e uso da tomografia de emissão de pósitron marcado com 18-fluorodesoxiglicose (PET-FDG) gerou questões sobre sua real importância para nosso metabolismo. Nesta revisão, discutiremos o que aprendemos nesse tempo, assim como conceitos antigos e novos, alguns marginalizados por décadas, como a termogênese induzida por dieta. Arq Bras Endocrinol Metab. 2014;58(9):889-99.

Limits and possibilities experienced by nurses in the treatment of women with chronic venous ulcers / Límites y posibilidades vividas por enfermeras en el tratamiento de mujeres con úlcera venosa crónica / Limites e possibilidades vivenciados por enfermeiras no tratamento de mulheres com úlcera venosa crônica

Objective To understand the experiences and expectations of nurses in the treatment of women with chronic venous ulcers. Method Phenomenological research was based on Alfred Schütz, whose statements were obtained in January, 2012, through semi-structured interviews with seven nurses. Results The nurse reveals the difficulties presented by the woman in performing self-care, the perceived limitations in the treatment anchored in motivation, and the values and beliefs of women. It showed professional frustration because venous leg ulcer recurrence, lack of inputs, interdisciplinary work and training of nursing staff. There was an expected adherence to the treatment of women, and it emphasized the need for ongoing care, supported self-care and standard practices in treatment. Conclusion That treatment of chronic venous leg ulcers constitutes a challenge that requires collective investment, involving women, professionals, managers and health institutions. .

Objetivo Comprender las experiencias y expectativas de enfermeras en el tratamiento de mujeres con úlcera venosa crónica. Método Investigación fenomenológica fundamentada en Alfred Schutz, que buscó Se realizó entrevista semiestructurada con siete enfermeras, en enero del 2012. Resultados La enfermera revela dificultades presentadas por la mujer para realizar el autocuidado, percibe limitaciones en el tratamiento relacionadas con la desmotivación, los valores y las creencias de las mujeres. Refiere frustración profesional debido a la recidiva de la lesión, a la falta de insumos, al deficiente trabajo interdisciplinar y a la limitada capacitación del equipo de enfermeras. Espera la adhesión de la mujer al tratamiento y resalta la necesidad del cuidado continuo, del autocuidado apoyado y de estandarizar conductas de tratamiento. Conclusión El tratamiento de la úlcera venosa crónica es un desafío que requiere contribución colectiva, involucrando a las mujeres, a los profesionales, a los gestores y a las instituciones de salud. .

Objetivo Compreender as experiências e expectativas de enfermeiras no tratamento de mulheres com úlcera venosa crônica na Atenção Primária à Saúde. Método Pesquisa fundamentada na fenomenologia social de Alfred Schütz, com depoimentos obtidos em janeiro de 2012, por meio de entrevista semiestruturada com sete enfermeiras. Resultados As enfermeiras revelam dificuldades apresentadas pelas mulheres com úlcera venosa crônica para realizar o autocuidado, percebem limitações na terapêutica ancoradas na desmotivação e nos valores e crenças das mulheres. Referem frustração profissional em razão da recidiva da lesão, falta de insumos e tecnologia, de trabalho interdisciplinar e da capacitação da equipe de enfermagem. Esperam a adesão das mulheres ao tratamento e ressaltam a necessidade do cuidado contínuo, do autocuidado apoiado e da padronização de condutas no tratamento. Conclusão O tratamento da úlcera venosa crônica constitui-se em um desafio que requer investimento coletivo, envolvendo a mulher, os profissionais, os gestores e as instituições de saúde. .

Pathophysiological insights in sickle cell disease.

The first coherent pathophysiological scheme for sickle cell disease (SCD) emerged in the sixties-seventies based on an extremely detailed description of the molecular mechanism by which HbS in its deoxy-form polymerises and forms long fibres within the red blood cell that deform it and make it fragile. This scheme explains the haemolytic anaemia, and the mechanistic aspects of the vaso-occlusive crises (VOCs), but, even though it constitutes the basic mechanism of the disease, it does not account for the processes that actually trigger VOCs. This paper reviews recent data which imply: red blood cell dehydration, its abnormal adhesion properties to the endothelium, the participation of inflammatory phenomenon and of a global activation of all the cells present in the vessel, and finally, abnormalities of the vascular tone and of nitric oxide metabolism. These data altogether have shed a new light on the pathophysiology of the first molecular disease i.e. sickle cell disease.

Activation of PPARgamma induces profound multilocularization of adipocytes in adult mouse white adipose tissues

We sought to determine the effects of activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) on multilocularization of adipocytes in adult white adipose tissue (WAT). Male C57BL/6 normal, db/db, and ob/ob mice were treated with agonists of PPAR-gamma, PPAR-alpha, or beta3-adrenoceptor for 3 weeks. To distinguish multilocular adipocytes from unilocular adipocytes, whole-mounted adipose tissues were co-immunostained for perilipin and collagen IV. PPAR-gamma activation with rosiglitazone or pioglitazone induced a profound change of unilocular adipocytes into smaller, multilocular adipocytes in adult WAT in a time-dependent, dose-dependent, and reversible manner. PPAR-alpha activation with fenofibrate did not affect the number of locules or remodeling. db/db and ob/ob obese mice exhibited less multilocularization in response to PPAR-gamma activation compared to normal mice. Nevertheless, all adipocytes activated by PPAR-gamma contained a single nucleus regardless of locule number. Multilocular adipocytes induced by PPAR-gamma activation contained substantially increased mitochondrial content and enhanced expression of uncoupling protein-1, PPAR-gamma coactivator-1-alpha , and perilipin. Taken together, PPAR-gamma activation induces profound multilocularization and enhanced mitochondrial biogenesis in the adipocytes of adult WAT. These changes may affect the overall function of WAT.

Role of ion channel modifiers in reversal of morphine-induced gastrointestinal inertia by prokinetic agents in mice.

Prokinetic drugs like mosapride, domperidone etc, are used to treat gastrointestinal delay. Though the receptor-mediated actions of these agents have been studied, involvement of ion channels in reversing morphine-induced gastrointestinal inertia by prokinetic agents has not been explored. Charcoal meal test was used to measure small intestinal transit (SIT) in adult male Swiss albino mice. Animals were given ion channel modifiers and prokinetic drugs intragastrically. Reversal of morphine-induced gastrointestinal delay by mosapride was decreased significantly by CaCl2, minoxidil and glibenclamide. Similarly, domperidone's effect on morphine was decreased by CaCl2, nifedipine, minoxidil and glibenclamide significantly. The results reveal that ion channel modifiers counteract the prokinetic effects of mosapride or domperidone.

Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 µM), or by inhibiting the H+-pump with bafilomycin (4 µM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 µM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations.

1a, 25(OH)2D3 induces capacitative calcium entry involving a TRPC3 protein in skeletal muscle and osteoblastic cells

This work describes the involvement of TRPC proteins in capacitative calcium entry (CCE) induced by 1a,25-dihydroxy-vitamin-D3 [1a,25(OH)2D3] in chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8) and the role of the vitamin D receptor (VDR) in this non-genomic rapid response mediated by the hormone. We propose that an endogenous TRPC3 protein mediates 1a,25(OH)2D3 modulation of CCE in these cells, which seems to implicate VDR-TRPC3 association and the participation of an INAD-like scaffold protein.

Dynamical complexity and temporal plasticity in pancreatic beta-cells.

We discuss some of the biological and mathematical issues involved in understanding and modelling the bursting electrical activity in pancreatic beta-cells. These issues include single-cell versus islet behaviour, parameter heterogeneity, channel noise, the effects of hormones, neurotransmitters, and ions, and multiple slow biophysical processes. Some of the key experimental and modelling studies are described, and some of the major open questions are discussed.

Natural probes for cholinergic sites: L-bebeerine actions on the neuromuscular transmission, the nicotinic receptor/ionic channel complex, and contraction of skeletal muscles

The mechanisms underlying the muscle relaxant of 1-bebeerine (BB), a tertiary alkaloid isolated from the roots of Chondrodendron platyphyllum, were examined in mammalian and amphibian skeletal muscles. Injections of BB (0.05 - 1 g/kg,i.p.) in rats caused a dose-related flaccid paralysis and respiratory arrest at high doses. In isolated rat diaphragmand toad sartorius muscles, BB depressed the indirectly elicited muscles twitches (IC50:228 muM and 5.4 muM, respectively, at 22 degree) and blocked the nerve-elicited muscle action potential. The neuromuscular blockade was not reserved by neostigmine (10 muM). High concentrations of BB (170 and 340 muM) caused muscle contracture unrelated to the junctional blockade, and intensified by increasing the bath temperature. Analysis of the contraction properties showed that BB (40 and 80 muM)increaded the twitch/tetanus ratio (46 percent and 125 percent) and prolonged the relaxation time; the falling phase of the directly elicited action potential in toad sartorius muscle fibers was slower probably by a decreased potasium conductance. BB (0.1 - 340 muM) reduced the binding of [1251]alpha- -bungarotoxin to the junctional AACh receptor of the rat diaphragm (IC50:47.7 muM, at 37 degree. At low concentrations BB (1.5 - 15 muM) induced either opening or blockade of the Ach receptor-ionic channel. The results showed that BB blocked noncompetitively the neuromuscular transmission through a mechanism that affects the Ach recognition site and the ionic channel properties. The alkaloid also produced muscle contracture and changed the contractile properties through its extra-junctional action at the calcium handling by the sarcoplasmic reticulum or the contractile machinery.

Patch-clamp detection of macromolecular translation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal `gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus.

Morphogenesis of the epithelial cell transporting phenotype: systhesis and distribution of ion channels

The exchange of substance between higher organisms and the environment takes place across epithelia consisting of one or more cell layers. To perform this function, epithelial cells have two basic differentiated properties: 1) they form tight junctions (Tjs) that seal the extracellular space, and 2) they are polarized into an apical and a basolateral domain, with entirely different structural, biochemical and physiological properties. Our understanding of the mechanisms involved in the expression of these properties has been greatly enchanced by the availability of epithelial cell lines that form Tjs and polarize in vitro under conditions suitable for experimental control. In this article we summarize our studies on the synthesis and polarized expression of ion channels in epithelial cells. MDCK cells have four types of K+ channels in the apical domain, and a fifth one in the basolateral domain. The basolateral side also has a population of Cl- channels. Each type of channel is absolutely polarized. Harvesting with trypsin-EDTA reduces the area of the plasma membrane by 50 per cent and the channel population by 90 per cent. Upon plating, these channels are recovered within a few hours. We describe here the main extracellular and intracellular mechanisms involved in these phenomena.

Noncompetitive inhibitors of the acetylcholine receptor help paint a picture of its ion channel

This review describes and analyzes the evidence from studies using noncompetitive inhibitors of the nicotinic acetylcholine receptor that the receptor's ion channel is formed by the second transmembrane segment of all five receptor subunits. Inconsistencies in this generally accepted model are also presented and discussed

Transporte de eletrolitos no epitelio respiratorio dos portadores de fibrose cistica / Eletrolyte transport by airway epithelia in patients with cystic fibrosis

Nas vias aereas de conducao, o transporte de ions tem papel importante na composicao das secrecoes. O canal de 'Cl POT -' e o canal de 'Na POT +' existentes na membrana apical controlam a secrecao e absorcao das celulas epiteliais. Fibrose cistica, o defeito fundamental e a regulacao do canal de'Cl POT -'. Estudos complementares ao nivel de regulacao molecular deste canal poderao levar ao melhor entendimento da doenca e as novas abordagens terapeuticas.