五种与癫癎治疗靶点相关蛋白质在颞叶癫癎模型海马组织中的表达

目的寻找颞叶癫大鼠海马组织的差异表达蛋白质,为寻找新的癫治疗靶点和研发新的治疗手段打下基础。方法运用二维电泳和MALD I-TOF-MS技术,对比分析氯化锂-匹罗卡品致大鼠海马组织和正常大鼠海马组织的蛋白质表达谱,对发现的差异表达蛋白质进行分析和鉴定。结果在氯化锂-匹罗卡品致大鼠海马组织中筛选到32个差异表达蛋白质斑点,其中20个在癫组表达下调,12个在癫组表达上调,其中5个蛋白质已被最终鉴定确认,分别为:突触结合蛋白Ⅰ(synaptotagm inⅠ)、神经丝蛋白(neurofilam ents,NF)、热休克蛋白27(heat shock prote in 27,HSP27)、电压依赖性阴离子通道1(voltage-dependent an ion channel prote ins 1,VDAC1)和异柠檬酸脱氢酶(isoc itric dehydrogenase,ICD),其中突触结合蛋白Ⅰ可能为潜在的新治疗靶点。结论颞叶癫大鼠海马组织中存在大量差异表达蛋白质,部分可能为潜在的癫治疗靶点。     

脑血管痉挛的差异蛋白质组学研究

目的采用蛋白质组学技术分离和鉴定动脉瘤性蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)患者组及无痉挛患者组发病后第1 d脑脊液中差异表达蛋白质,寻找与脑血管痉挛相关的蛋白质。方法采用双向凝胶电泳分离无脑血管痉挛组(对照组)、轻中度痉挛组(实验组1)和重度痉挛组(实验组2)脑脊液总蛋白质,比较分析实验组和对照组之间的差异表达蛋白质,应用基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF/TOF-MS)鉴定差异表达的蛋白质。结果实验组1、实验组2与对照组表达差异2倍以上的蛋白质斑点共有68个,成功鉴定出23个差异表达蛋白质。结论初步建立了脑血管痉挛的差异表达蛋白质双向电泳技术体系,筛选出一批与血管痉挛相关蛋白质候选物。本研究为脑血管痉挛的发病机制研究提供了新的方法,为寻找脑血管痉挛的生物标志物、寻找药物治疗靶标和预后评估打下研究基础。     

人脑星形胶质细胞瘤恶性程度相关蛋白表达谱的分析与鉴定

目的 应用蛋白质组学技术确定与人脑星形胶质细胞瘤恶性程度相关的差异表达蛋白质分子. 方法 二维电泳技术比较分析12例正常脑组织和52例不同恶性程度星形胶质细胞瘤组织(Ⅱ级23例、Ⅲ级15例、Ⅳ级14例)差异表达蛋白质斑点,高效液相色谱-电喷雾(ESI)串联质谱技术、生物信息学方法分析鉴定与恶性程度相关的差异表达蛋白质分子. 结果 共鉴定出15种差异表达的蛋白质分子,其中热休克蛋白27、抑制素、葡萄糖调节蛋白78、过氧化物氧化酶1、过氧化物氧化酶6、膜联蛋白Ⅱ、组织蛋白酶D、纤溶酶原激活物抑制剂-1、肌动蛋白(胞浆型1)、谷胱甘肽S-转移酶M这10种蛋白的表达在Ⅲ级和Ⅳ级星形胶质细胞瘤组织中升高,二硫键异构酶A3、αB晶体蛋白质、T复合体蛋白1(ε亚单位)、泛素C-末端水解酶同T酶L1、胶质纤维酸性蛋白这5种蛋白的表达降低. 结论 蛋白质组学技术可有效鉴定与肿瘤恶性程度相关的差异表达蛋白,为星形胶质细胞瘤的分子个体诊断和分子靶向治疗提供实验依据.     

蛋白质组学技术鉴定帕金森病模型中的3种新蛋白质

目的 利用蛋白质组学技术对MPP 诱导的PC12细胞帕金森病(PD)模型进行研究,以期发现PD发病机制中的新蛋白.方法 建立MPP 诱导的PC12细胞PD模型并提取细胞总蛋白,荧光差异凝胶电泳(D1GE)进行蛋白样品标记与分离,应用DeCyder软件分析蛋白质差异表达信息,运用MALDI-TOF质谱鉴定差异蛋白质.结果 质谱分析和数据库检索鉴定了3种可能同PD发病相关但在有关PD研究文献中尚未见报道的新蛋白质,他们分别足与线粒体功能相关的蛋白MPPs,具有分子伴侣活性的蛋白NAC和与免疫炎症相关的蛋白gc1qBP.结论 这些新发现蛋白可能与PD的发病机制密切相关.     

NGF诱导PC12细胞早期分化的DIGE分析

目的筛选神经生长因子(Nerve growth factor,NGF)处理后PC12细胞分化早期相关蛋白,寻找NGF药物作用靶点,为深入进行神经保护药物的研发提供理论依据。方法在建立NGF诱导PC12细胞分化模型的基础上,分别提取NGF处理前后的细胞总蛋白,应用荧光差异凝胶电泳(Differential Gel Electrophoresis,DIGE)系统获得蛋白点表达差异信息,运用MALDI-TOF质谱鉴定出差异蛋白质。结果NGF处理组有7个蛋白点发生变化;通过数据库检索,鉴定了3种结构和功能各异的蛋白。结论eEF-2、Rab GDIα和DPP与NGF诱导PC12细胞分化早期过程有关,可能是NGF的作用靶点。     

癫痫患者脑脊液差异蛋白质组学分析

目的应用蛋白质组学技术研究脑脊液的癫痫相关蛋白,以便从蛋白质水平揭示癫痫发生发展机制及可能的诊断标志物。方法双向凝胶电泳和质谱技术分离和鉴定癫痫组(n=8)和对照组(n=8)脑脊液的差异表达蛋白。结果约68个蛋白质点表达水平在两组间存在明显差异,选择18个差异蛋白质点质谱鉴定。除4个假想蛋白和未知功能蛋白及得到7个已知功能蛋白,涉及免疫反应、运输蛋白、能量代谢酶蛋白、细胞骨架蛋白和热休克蛋白。结论大部分被鉴定的蛋白为首次报道,这些蛋白与癫痫的发生发展相关,可能成为癫痫脑脊液分子标志物。     

骨髓源神经干细胞移植治疗中枢神经系统损伤的蛋白质组学研究

目的 利用同位素标记相对和绝对定量(iTRAQ)技术以及串联质谱技术分析中枢神经系统损伤患者干细胞移植前术后脑脊液蛋白质表达变化情况.方法 收集正常成人以及中枢神经系统损伤患者移植前后脑脊液,组内等量混合后利用高效液相色谱柱去除高丰度蛋白,样本经iTRAQ试剂标记后利用二维色谱分离,经串联质谱鉴定并相对定量.结果 质谱得到的多肽片段经谱库搜索鉴定的蛋白共59个,其中患者移植前脑脊液与正常对照组脑脊液存在差异的蛋白质有13个,5个呈高表达,8个呈低表达.患者移植后脑脊液有7种蛋白质与正常对照组表达无差异.结论 蛋白质组学研究方法可以用来寻找神经系统损伤标志物,为神经系统损伤以及修复的机制研究提供基础.     

卡莫司汀致皮质发育障碍的模型研究

目的 建立SD大鼠广泛型皮质发育障碍的动物模型.方法 在SD大鼠孕17 d腹腔注入BCNU(1,3-二氯乙烯一亚硝基脲,卡莫司汀)制作皮质发育障碍模型;病理检查P60仔鼠皮层和海马结构及神经元变化;行为学观察和脑电图检测;热水浴诱导痫性发作,观察两组大鼠的致痫潜伏期和癫痫持续时间;Y迷宫法测试不同时间点仔鼠学习记忆能力.结果 PO仔鼠脑组织湿重实验组比对照组显著减轻(P<0.01);Nissl染色显示皮质层次紊乱、海马区域异位细胞异常聚集,可出现结节状灰质团块;模型鼠日常活动能力较差,脑电图未见明显痫性放电;模型组热水浴诱导惊厥发作的潜伏期明显缩短(P<0.01);模型组达到学会标准所需电击次数较对照组增加(P<0.05).结论 孕17 d腹腔注射BCNU可建立广泛型皮质发育障碍的动物模型,其致痫敏感性增加,且伴有认知功能障碍.     

颅脑创伤后亚低温脑保护的蛋白质组研究

目的 本研究应用差异蛋白质组学技术,探讨亚低温和常温条件下,创伤性颅脑损伤大鼠海马组织蛋白质的表达变化.方法 采用侧方液压冲击装置,建立大鼠中度脑损伤模型,亚低温组(n=3)于伤后维持体温(33±0.5)℃持续3h,常温组(n=3)始终维持体温(37±0.5)℃.取大鼠海马组织,通过差异荧光双向凝胶电泳、分离蛋白,获得二维的蛋白质分离图谱,然后通过胶内酶切、抽提酶解肽段、基质辅助激光解吸飞行时间质谱分析差异的蛋白质点,鉴定出变化的蛋白质.结果 通过DeCyder5.0(GE Healthcare)软件分析报告发现差异1.5倍以上的蛋白点17个(P＜0.05).通过对这些蛋白考染点进行质谱鉴定,鉴定出14个蛋白质,2个为同一种蛋白,实际差异蛋白数为13个.分别是细胞骨架蛋白、介导能量代谢的酶类、参与核酸合成、氧化应激反应的蛋白质、神经突触功能蛋白、细胞内信号传递蛋白及未知蛋白.结论 脑损伤后亚低温及常温条件下,大鼠海马组织蛋白质存在表达差异,鉴定出的差异蛋白质可能与亚低温保护效应的潜在作用机制有关.     

海人酸致痫大鼠海马及颞叶皮质区域NMDA受体亚单位R1蛋白的表达

背景：研究表明天门冬氨酸受体亚单位R1与癫痫的发生有关,但其具体的表达情况与癫痫脑损伤的关系尚不明确。 目的：观察海人酸致痫大鼠海马及颞叶皮质区域天门冬氨酸受体亚单位1蛋白表达的变化。 设计、时间及地点：随机对照动物实验,于2002-03/2003-03在吉林大学基础医学院生理学和病理生物学教研室完成。 材料：兔抗天门冬氨酸受体受体亚单位1多克隆抗体购自武汉博士德生物科技公司。 方法：将80只雄性健康鼠龄22周的Wistar大鼠随机分为模型组70只和假手术组10只。模型组注射海人酸1 μL至右侧杏仁核,制备癫痫动物模型,分别于造模后2,6,24,72 h及7,15,30 d各取10只动物处死。假手术组在大鼠右侧杏仁核注入PBS 1 μL。 主要观察指标：致痫后2,6,24,72 h及7,15,30 d应用流式细胞仪技术及免疫组织化学的方法观察大鼠脑组织海马及颞叶皮质天门冬氨酸受体受体亚单位1蛋白表达的变化。 结果：假手术组海马各区及颞叶皮质有极少量的天门冬氨酸受体受体亚单位1蛋白阳性细胞分布,海人酸致痫后2 h大鼠海马各区及颞叶皮质天门冬氨酸受体受体亚单位1蛋白表达迅速增加,6 h明显升高,7 d略有下降（CA3区及颞叶皮质）但仍高于假手术组,并持续至30 d（P<0.01）。致痫后海马CA3区、齿状回与颞叶皮质相比,NMDAR1阳性细胞数增多 (P < 0.01)。 结论：海人酸致痫后大鼠海马及颞叶皮质天门冬氨酸受体受体亚单位1蛋白的表达水平均上调,在海马CA3及齿状回较为明显,并持续至致痫后的30 d。天门冬氨酸受体受体亚单位1可能参与了癫痫发生和癫痫脑的长时程的兴奋过程。     

Disorders of cortical development and epilepsy

There has been an impressive increase in our ability to identify and categorize patients with cortical development lesions over the past decade. The clinical features associated with disorders of cortical development (DCD) have been described, and epilepsy has been shown to be a frequent symptom. In this review, we categorize DCD based on their structure and discuss their underlying causes and clinical features. Just as the cause of each type of disorder is thought to be unique, each disorder also has distinct types of seizures, treatment strategies, and electroencephalographic features. Studies in human tissue and animal models of DCD have begun to shed light on why DCD are associated with epilepsy. Aberrant synaptic connections within the dysplastic tissue and between the dysplastic tissue and more normal-appearing adjacent tissue form an abnormal, hyperexcitable network that increases seizure susceptibility. In the future, strategies for blocking formation of the aberrant networks may prevent the development of epilepsy.     

Changes in intracellular protein expression in cortex, thalamus and hippocampus in a genetic rat model of absence epilepsy

Epilepsy is a chronic disorder characterized by repeated seizures resulting from abnormal activation of neurons in the brain. Although mutations in genes related to Na⁺, K⁺, Ca²⁺ channels have been defined, few studies show intracellular protein changes. We have used proteomics to investigate the expression of soluble proteins in a genetic rat model of absence epilepsy “Genetic Absence Epilepsy Rats from Strasbourg (GAERS)”. The advantage of this technique is its high throughput quantitative and qualitative detection of all proteins with their post-translational modifications at a given time. The parietal cortex and thalamus, which are the regions responsible for the generation of absence seizures, and the hippocampus, which is not involved in this activity, were dissected from GAERS and from non-epileptic control rat brains. Proteins from each tissue sample were isolated and separated by two-dimensional gel electrophoresis. Spots that showed significantly different levels of expression between controls and GAERS were identified by nano LC-ESI-MS/MS. Identified proteins were: ATP synthase subunit delta and the 14-3-3 zeta isoform in parietal cortex; myelin basic protein and macrophage migration inhibitory factor in thalamus; and macrophage migration inhibitory factor and 0-beta 2 globulin in hippocampus. All protein expressions were up-regulated in GAERS except 0-beta globulin. These soluble proteins are related to energy generation, signal transduction, inflammatory processes and membrane conductance. These results indicate that not only membrane proteins but also cytoplasmic proteins may take place in the pathophysiology and can be therapeutic targets in absence epilepsy.     

Regional cerebral cortical thinning in bipolar disorder

OBJECTIVE: This study was conducted to explore differences in cortical thickness between subjects with bipolar disorder and healthy comparison subjects using cortical surface-based analysis. METHODS: Brain magnetic resonance images were acquired from 25 subjects with bipolar disorder and 21 healthy comparison subjects. Cortical surface-based analysis was conducted using the Freesurfer application. Group differences in cortical thickness, defined by the distance from gray/white boundary to the pial surface, were assessed using statistical difference maps. RESULTS: Subjects with bipolar disorder exhibited significantly decreased cortical thickness in left cingulate cortex, left middle frontal cortex, left middle occipital cortex, right medial frontal cortex, right angular cortex, right fusiform cortex and bilateral postcentral cortices, relative to healthy comparison subjects (all p < 0.001). Duration of illness in bipolar subjects was inversely correlated with the cortical thickness of the left middle frontal cortex. CONCLUSIONS: Cortical thinning was present in multiple prefrontal cortices in bipolar disorder. There was also cortical thinning in sensory and sensory association cortices, which has not been reported in previous studies using region-of-interest or voxel-based morphometry methods. Cortical thinning observed in the current study may be related to impairment of emotional, cognitive, and sensory processing in bipolar disorder but longitudinal studies will be necessary to test this hypothesis.     

Investigation of widespread neocortical pathology associated with hippocampal sclerosis in epilepsy: a postmortem study

Purpose: One possible cause for surgical failure following temporal lobectomy for the treatment of epilepsy due to classical hippocampal sclerosis (CHS) is the presence of more widespread cortical changes. Neocortical changes in CHS shown by quantitative neuroimaging studies may involve hippocampal projection pathways. Our aim was to quantitate neocortical pathology using a postmortem series of brains from patients with epilepsy and CHS. Methods: Sections from 13 cortical regions from both left and right hemispheres, including hippocampal projection pathways, were examined from nine epilepsy patients with unilateral CHS (4), bilateral CHS (2), non‐CHS (3), and non–epilepsy controls (4). Using GFAP, CD68, and NPY immunohistochemistry as markers of acquired neocortical pathology, quantitative analysis of the staining fractions in the cortex and white matter was carried out. Key Findings: Higher staining fractions were observed for all markers in both cortex and white matter in CHS patients, which was significantly different for CD68 and NPY compared to controls (p < 0.05) but not to non‐CHS epilepsy cases. There was no significant difference between staining fractions in left and right hemispheres for unilateral CHS cases. Regional analysis showed preferential gliosis and microgliosis of temporal poles, frontal poles, and orbitofrontal cortex in epilepsy cases. Significance: This study supports acquired neocortical pathology in epilepsy patients both with and without CHS. Cortical pathology does not show lateralization to the side of CHS. Preferential involvement of the temporal and frontal poles may relate to other factors, such as cortical injury associated with seizures, rather than involvement through hippocampal pathways.     

Regional increase of cerebral cortex thickness in juvenile myoclonic epilepsy

The goal of this study was to characterize cerebral cortex thickness patterns in juvenile myoclonic epilepsy (JME). Surface‐based morphometry (SBM) was applied to process brain magnetic resonance images acquired from 24 patients with JME and 40 healthy controls and quantify cerebral cortex thickness. Differences in cortical thickness between patients and controls were determined using generalized linear model (covariates: age and gender). In patients with JME, thickness increase was detected bilaterally within localized regions in the orbitofrontal and mesial frontal cortices. Such thickness patterns coexisted with significant bilateral reduction in thalamic volume. These findings confirm that the underlying mechanisms in JME are related to aberrant corticothalamic structure and indicate that frontal cortex abnormalities are possibly linked to regional increase in cerebral cortical thickness.     

Increased expression of urokinase‐type plasminogen activator receptor in the frontal cortex of patients with intractable frontal lobe epilepsy

Urokinase‐type plasminogen activator receptor (uPAR) is a glycosyl phosphatidylinositol‐anchored protein involved in cell adhesion, proliferation, differentiation, migration, invasion, and tissue repair and remodeling. Our aim was to investigate uPAR expression in the frontal cortex of patients with intractable frontal lobe epilepsy and to explore the possible role of uPAR in intractable epilepsy. Tissue samples were obtained from the frontal cortex of 25 patients who had undergone surgery for intractable epilepsy and 15 histologically normal frontal cortex tissues from patients with orbital frontal lobe severe contusion (the control group). The frontal cortex expression of uPAR was studied by Western blot and immnohistochemistry. Double immunofluorescence was used to determine the expression of uPAR in astrocytes, microglia, and neurons. The normal frontal cortex uPAR protein level was shown to be low. In the brain tissue of patients with intractable epilepsy, the expression of uPAR protein increased dramatically. Based on the results of double immunofluorescence, many uPAR‐positive cells are colocalized with the cell soma of NeuN‐positive neurons, whereas only a few GFAP‐ and CD11b‐positive cells colocalized with uPAR staining. These findings provide new information pertaining to the epileptogenesis of intractable epilepsy and suggest that increased expression of uPAR in human brain may be associated with human intractable epilepsy. © 2010 Wiley‐Liss, Inc.     

Reduced relative volume in motor and attention regions in developmental coordination disorder: A voxel-based morphometry study

Background and objectivesDevelopmental coordination disorder (DCD) is a prevalent childhood movement disorder, impacting the ability to perform movement skills at an age appropriate level. Although differences in grey matter (GM) volumes have been found in related developmental disorders, no such evidence has been linked with DCD to date. This cross-sectional study assessed structural brain differences in children with and without DCD.MethodsHigh-resolution structural images were acquired from 44 children aged 7.8–12 years, including 22 children with DCD (≤16th percentile on MABC-2; no ADHD/ASD), and 22 typically developing controls (≥20th percentile on MABC-2). Structural voxel-based morphology analysis was performed to determine group differences in focal GM volumes.ResultsChildren with DCD were found to have significant, large, right lateralised reductions in grey matter volume in the medial and middle frontal, and superior frontal gyri compared to controls. The addition of motor proficiency as a covariate explained the between-group GM volume differences, suggesting that GM volumes in motor regions are reflective of the level of motor proficiency. A positive correlation between motor proficiency and relative GM volume was also identified in the left posterior cingulate and precuneus.ConclusionsGM volume reductions in premotor frontal regions may underlie the motor difficulties characteristic of DCD. It is possible that intervention approaches targeting motor planning, attention, and executive functioning processes associated with the regions of reduced GM volume may result in functional improvements in children with DCD.     

Up-regulation of major vault protein in the frontal cortex of patients with intractable frontal lobe epilepsy

Major vault protein (MVP) is a vesicular drug transporter and may participate in multidrug resistance (MDR). The aim of this study was to determine the expression and cellular localization of MVP in refractory frontal lobe epilepsy (FLE). We detected MVP expression in tissue samples from the refractory frontal cortex of 30 patients who had been surgically treated for refractory epilepsy. We compared these tissues with twelve histologically normal frontal lobe samples from controls. In the control group, the expression of MVP was faint in the cortex. The expression of MVP protein increased dramatically in the refractory epilepsy group; MVP immunoreactivity (IR) was observed in the cytoplasm of neurons. Thus, MVP protein was increased in the frontal cortex of patients with refractory epilepsy. Further research is necessary to determine whether or not MVP plays a role in the mechanisms underlying drug resistance in refractory FLE.     

Widespread cortical thinning in children with frontal lobe epilepsy

Purpose: Spread of seizure activity outside the frontal lobe due to cortico‐cortical connections can result in alteration in the cortex beyond the frontal lobe in children with intractable frontal lobe epilepsy (FLE). The aim of this study was to identify regions of reduced cortical thickness in children with intractable FLE. Methods: High‐resolution volumetric T₁‐weighted imaging was performed on 17 children with FLE, who were being evaluated for epilepsy surgery, and 26 age‐matched healthy controls. The cortical thickness of 12 patients with left FLE and 5 patients with right FLE was compared to controls. The clusters of cortical thinning were regressed against age of seizure onset, duration of epilepsy, seizure frequency, and number of medications. Key Findings: In children with left FLE, cortical thinning was present in the left superior frontal, paracentral, precuneus, cingulate, inferior parietal, supramarginal, postcentral, and superior temporal gyri, as well as in the right superior and middle frontal, medial orbitofrontal, supramarginal, postcentral, banks of superior temporal sulcus, and parahippocampal gyri. In children with right FLE, cortical thinning was present in the right precentral, postcentral, transverse temporal, parahippocampal, lingual, and lateral occipital gyri, as well as in the left superior frontal, inferior parietal, postcentral, superior temporal, posterior cingulate, and lingual gyri. In children with left FLE, following exclusion of one outlier, there was no significant association between age at seizure onset, duration of epilepsy, seizure frequency and number of medications with clusters of cortical thinning. In children with right FLE, age at seizure onset, duration of epilepsy, frequency of seizures, and number of medications were not associated with clusters of cortical thinning within the right and left hemispheres. Significance: Cortical changes were present in the frontal and extrafrontal cortex in children with intractable FLE. These changes may be related to spread of seizure activity, large epileptogenic zones involving both frontal and extrafrontal lobes, and development of secondary epileptogenic zones that over time lead to cortical abnormality. Further studies correlating cortical changes with neurocognitive measures are needed to determine if the cortical changes relate to cognitive function.     

Simultaneous EEG,fMRI, and behavior in typical childhood absence seizures

Purpose: Absence seizures cause transient impairment of consciousness. Typical absence seizures occur in children, and are accompanied by 3–4‐Hz spike–wave discharges (SWDs) on electroencephalography (EEG). Prior EEG–functional magnetic resonance imaging (fMRI) studies of SWDs have shown a network of cortical and subcortical changes during these electrical events. However, fMRI during typical childhood absence seizures with confirmed impaired consciousness has not been previously investigated. Methods: We performed EEG‐fMRI with simultaneous behavioral testing in 37 children with typical childhood absence epilepsy (CAE). Attentional vigilance was evaluated by a continuous performance task (CPT), and simpler motor performance was evaluated by a repetitive tapping task (RTT). Results: SWD episodes were obtained during fMRI scanning from 9 patients among the 37 studied. fMRI signal increases during SWDs were observed in the thalamus, frontal cortex, primary visual, auditory, somatosensory, and motor cortex, and fMRI decreases were seen in the lateral and medial parietal cortex, cingulate gyrus, and basal ganglia. Omission error rate (missed targets) with SWDs during fMRI was 81% on CPT and 39% on RTT. For those seizure epochs during which CPT performance was impaired, fMRI changes were seen in cortical and subcortical structures typically involved in SWDs, whereas minimal changes were observed for the few epochs during which performance was spared. Discussion: These findings suggest that typical absence seizures involve a network of cortical–subcortical areas necessary for normal attention and primary information processing. Identification of this network may improve understanding of cognitive impairments in CAE, and may help guide development of new therapies for this disorder.