Cell type-dependent, infection-induced, aberrant DNA methylation in gastric cancer

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders. In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed. In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.

Genetically engineered sensors of cell signaling

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.

Efeitos da hipóxia-isquemia perinatal sobre o comportamento motor, distribuição da Tirosina Hidroxilase na substância negra e da NADPH diaforase no hipocampo durante o desenvolvimento em ratos

A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento.

Transcriptome of embryonic and neonatal mouse cortex by high-throughput RNA sequencing

Brain structure and function experience dramatic changes from embryonic to postnatal development. Microarray analyses have detected differential gene expression at different stages and in disease models, but gene expression information during early brain development is limited. We have generated >27 million reads to identify mRNAs from the mouse cortex for>16,000 genes at either embryonic day 18 (E18) or postnatal day 7 (P7), a period of significant synapto-genesis for neural circuit formation. In addition, we devised strategies to detect alternative splice forms and uncovered more splice variants. We observed differential expression of 3,758 genes between the 2 stages, many with known functions or predicted to be important for neural development. Neurogenesis-related genes, such as those encoding Sox4, Sox11, and zinc-finger proteins, were more highly expressed at E18 than at P7. In contrast, the genes encoding synaptic proteins such as synaptotagmin, complexin 2, and syntaxin were up-regulated from E18 to P7. We also found that several neurological disorder-related genes were highly expressed at E18. Our transcriptome analysis may serve as a blueprint for gene expression pattern and provide functional clues of previously unknown genes and disease-related genes during early brain development.

Developmental expression of CagMdkb during gibel carp embryogenesis

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.

非洲爪蟾早期发育中的转录后调控机制及眼睛发育相关基因的表达研究

近来的研究表明，转录后调控对于调节脊椎动物发育过程中的细胞分化，细胞分裂及基因区域特异性表达都具有重要作用。转录后调控包括对mRNA稳定性、翻译效率、细胞内定位及poly(A)水平的调控等。Sox2基因是脊椎动物早期发育中最早表达的神经系统特异性基因之一，是脊椎动物早期神经系统发育的重要调节因子。通过生物信息学分析，我们发现，在脊椎动物Sox2 mRNA 3’非翻译区中存在4段非常保守的富含AU的区域，通过报告基因分析等手段研究发现Sox2 3’非翻译区中的部分元件可显著提高报告基因表达，提示我们Sox2的表达可能受到转录后调控。 我们通过对爪蟾Xfhl3基因序列分析时发现其3’非翻译区存在一段保守的只在两栖类具有的序列，我们克隆并检测了该基因的表达图式，并采用报告基因分析等手段研究了Xfhl3基因 3’非翻译区对报告基因表达的影响。结果发现其3’UTR可抑制报告基因表达水平。由于Xfhl3基因3’UTR中这段序列只在爪蟾基因中高度保守，而在在进化过程中两栖动物最独特的便是变态现象，这提示我们去探索这段爪蟾特有的保守序列是否与两栖类变态发育密切相关。由于甲状腺激素在两栖类的变态中的重要作用，因此我们设想Xfhl3基因的3’UTR中的保守序列可能与甲状腺激素相互作用共同调节爪蟾的变态过程。我们的初步结果表明，在爪蟾胚胎中，甲状腺激素对于正常报告基因表达没有明显的作用，但是在插入Xfhl3基因3’UTR中保守序列后，甲状腺激素处理可显著提高报告基因的表达，表明甲状腺素可能直接或间接通过与该段保守序列参与基因的表达调控。 脊椎动物的眼是一个功能非常特殊的器官，受到复杂的调控网络的调节，众多对神经发生重要的基因在眼中表达并参与了这一调节过程。我们克隆了非洲爪蟾的Sox1基因并研究了它在非洲爪蟾早期发育过程中的时空表达图式，比较了Sox1-3基因在发育的脑和眼中的表达图式，进一步阐明SoxB1基因家族在脊椎动物神经系统发生过程中的作用。此外，我们还克隆了非洲爪蟾MGC85160基因并利用RT-PCR和胚胎整体原位杂交技术探测它在不同胚胎阶段的时空表达图式。结果表明母源性表达的MGC85160基因早期主要在动物极表达；从神经板期开始在发育的中枢神经系统和眼中表达，石蜡切片显示它主要在视网膜和晶状体中表达，说明该基因在爪蟾早期外胚层的模式化以及中枢神经系统的发育过程中可能起到重要作用。 此外，我们还研究了鱇浪白鱼的早期发育分期和眼睛特异基因的表达图式。鱇浪白鱼（Anabarilius grahami ）是云南抚仙湖的特有鱼种。我们首次完成了鱇浪白鱼早期发育的完整分期，主要包括合子期，卵裂期，囊胚期，原肠期，体节期和孵化期六个主要的时期。为了理解鱇浪白鱼眼睛的发育，我们克隆并检测了在眼发育早期起关键作用的基因Sox2, Pax6a, Six3a 和 Rx2的表达图式。结果表明这四个基因全部在尾芽期的前端神经板中表达，随后在视网膜原基细胞中表达明显。在晚期阶段，除Rx2外其它三个基因也在晶状体中表达。其表达模式与斑马鱼中同源基因的表达很相似，说明涉及眼发育的分子网络在鱇浪白鱼中也是高度保守的。

非洲爪蛙分泌型叶酸结合蛋白及NKX6.3在早期胚胎神经发育中的功能研究

叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此我们认为sFBP结合叶酸后可能通过细胞膜上的受体传递信号，并且Islet-1可能在sFBP的下游发挥作用。 神经嵴是脊椎动物特有的一群多潜能干细胞，产生于表皮和神经板的边界，在原肠运动之后这群细胞通过表皮间充值转换从神经管背侧迁移到不同的区域，分化成不同的细胞类型，包括外周神经系统，色素细胞，软骨等。神经嵴的发生是一个多步骤多基因参与的精细调控过程。目前理论认为最初由一些分泌性信号分子又叫形态生成素比如BMP,Wnt，FGF，Notch等通过不同浓度梯度的相互作用调节一组在表皮和神经板边界的转录因子（Msx、Pax3/7、Zic1、Dlx3/5等）的表达，即边界决定。这些边界决定因子进一步在预定形成神经嵴的区域激活神经嵴特化基因比如Slug/Snail、FoxD3、Twist、Sox9/10的表达完成神经嵴的特化（Specification）。 Nkx6.3是Nkx6家族的一个转录因子，RT-PCR显示其呈现母源性表达。特异抗体显示Nkx6.3蛋白第9期在整个胚胎都表达，大部分蛋白集中在细胞核，有少部分蛋白定位于细胞膜上；神经板时期主要定位于神经嵴区域的细胞膜上。过表达Nkx6.3会影响细胞粘连分子的表达，由此干扰正常的胚胎原肠运动和Activin诱导的动物帽聚集延伸运动。显微注射Nkx6.3特异morpholino阻断其蛋白表达会抑制神经嵴的marker基因Wnt8，Fgf8，Pax3，Msx1，Zic1，FoxD3，Slug的转录，阻碍神经嵴的发育。在动物帽中单独注射Nkx6.3可以在mRNA水平上诱导Wnt8、Fgf8另一方面抑制BMP4的表达进而诱导神经嵴基因Pax3，Zic1，Slug的表达。报告基因实验也显示Nkx6.3能够激活Wnt信号而在动物帽中抑制BMP信号。Nkx6.3蛋白功能域分析发现其EH1结构域（domain）参与对Wnt8信号的激活，而EH1结构域和HD结构域之间的连接区域（linker domain）参与对FGF的激活和对BMP的抑制。进一步在动物帽和胚胎中分析发现Nkx6.3对Wnt8的激活依赖于FGF家族受体信号但是不依赖于Fgf8。有趣的是4细胞时期过表达Nkx6.3促进Fgf8和Wnt8 mRNA表达，但是抑制边界决定基因Msx1、Pax3和神经嵴特化基因Slug的转录。在32细胞时期显微注射Nkx6.3可以在内源神经嵴发生区域抑制Slug的表达，而异位却诱导Slug的mRNA。我们发现与动物帽中对BMP的调节不同，在胚胎中，过表达Nkx6.3会强烈的激活Smad1蛋白在细胞核中的表达即BMP信号被激活，高的BMP信号会抑制神经嵴的发生。另外我们发现过表达Nkx6.3在胚胎中抑制Dlx5而在动物帽中却不影响Dlx5的表达水平，Morpholino阻断Dlx5会抑制Msx1、Pax3和Slug的表达。BMP信号和Dlx5在动物帽和在整体胚胎中对Nkx6.3的不同响应可以一定程度上解释过表达Nkx6.3在2个系统中对神经嵴基因Slug相反的影响结果。

鸡胚吗啡用药对学习记忆和成瘾易感性的影响及其受体机制

Prenatal morphine exposure affects neural development of fetus by impairing learning and memory, and increasing susceptibility to morphine abuse. Because nervous systems have different developmental characteristics during different developmental stages, administration of morphine at different stages also has different effects on learning, memory, and susceptibility to morphine. Due to the precise developmental processes of neurotransmitter systems in chick embryo’s brain, and unique superiority of chick embryo model, the purpose of the present studies was to explore critical periods correlated to the memory impairment and the increasing susceptibility to morphine, via one-trial passive avoidance and conditioned place preference as behavior models. Then the possible roles of mu and delta opioid receptors as the possible mechanism were analyzed. Experiment 1 showed that injecting low dose of morphine (1 mg/kg) during the period embryonic 5 to 8 significantly impaired the function of learning and memory, worse than any other periods of the same treatment. Experiment 2 showed that injecting low dose of morphine during the period embryonic 17 to 20 significantly increased the susceptibility to morphine in the new-born chicks. The affected chicks acquired the morphine conditioned place preference more quickly, and maintained it much longer. Experiment 3 showed that during E5-8, injecting delta receptor antagonist naltrindole reversed the learning and memory impairment caused by morphine while delta receptor agonist DPDPE impaired learning and partial memory function. On the other hand, mu opioid receptors had little effect. As for E17-20, given naloxonazine can reverse the increases of susceptibility to morphine, and the mu receptor agonist DAGO cause the increases of susceptibility to morphine. Delta receptors have no effect. The above results demonstrated that prenatal morphine expousure at different developmental periods of chick embryo caused different influences on memory and susceptibility to morphine. That is, E5-8 is the critical period correlate to memory impairment; and E17-20 is the critical period correlate to susceptibility to morphine. Delta receptors were critical in learning and memory impairment while mu receptors in susceptibility.

The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function

The conserved habenular neural circuit relays cognitive information from the forebrain into the ventral mid- and hindbrain. In zebrafish, the bilaterally formed habenulae in the dorsal diencephalon are made up of the asymmetric dorsal and symmetric ventral habenular nuclei, which are homologous to the medial and lateral nuclei respectively, in mammals. These structures have been implicated in various behaviors related to the serotonergic/dopaminergic neurotransmitter system. The dorsal habenulae develop adjacent to the medially positioned pineal complex. Their precursors differentiate into two main neuronal subpopulations which differ in size across brain hemispheres as signals from left-sided parapineal cells influence their differentiation program. Unlike the dorsal habenulae and despite their importance, the ventral habenulae have been poorly studied. It is not known which genetic programs underlie their development and why they are formed symmetrically, unlike the dorsal habenulae. A main reason for this lack of knowledge is that the vHb origin has remained elusive to date.

Controlo postural na sequência de movimento de sentado para de pé em crianças com alterações do desenvolvimento neuromotor

A Sequência de Movimento de Sentado para de Pé (SMSP) é um marco importante na independência funcional da criança, sendo a sua qualidade afetada em casos de alteração do controlo postural do tronco. É por isso motivo de especial foco neste estudo, como elemento avaliativo e interventivo. Este estudo tem como objetivo a análise da modificação de componentes motores relacionados com o controlo postural, durante a SMSP, em 5 crianças com alterações neuromotoras, face à aplicação de um programa de intervenção baseado no Tratamento de Neuro Desenvolvimento (TND). Foi para tal utilizada a análise cinemática da SMSP - nomeadamente as variáveis “variação do deslocamento dos segmentos cabeça e tronco” e “variação do ângulo perna-pé” entre o momento inicial e o seat-off – complementada com a aplicação do Teste de Medida da Função Motora 88 (TMFM-88) e a Classificação Internacional da Funcionalidade Incapacidade e Saúde-Versão Crianças e Jovens (CIF-CJ), bem como a avaliação de componentes de movimento com recurso a registo de imagem. A análise cinemática demonstrou uma diminuição do deslocamento dos segmentos cabeça e tronco na maioria dos casos, bem como uma maior mobilidade da tíbia sobre pé. Verificou-se um aumento do score final do TMFM-88, em todos os casos. A CIF-CJ não evidenciou alterações entre o início e o término do período de intervenção. O registo de imagem demonstrou alterações positivas visíveis no alinhamento de segmentos, nível de atividade, controlo postural, e recurso a estratégias compensatórias no conjunto postural sentado e na SMSP. Após o período de intervenção as crianças deste estudo evidenciaram modificações positivas nas variáveis em estudo, nomeadamente 1) diminuição do deslocamento dos segmentos cabeça e tronco na fase inicial da SMSP, 2) aumento da variação do ângulo perna-pé na fase inicial da SMSP, 3) aumento do score total da TMFM-88, 4) alteração positiva dos componentes de movimento alinhamento de segmentos e nível de atividade e 5) redução do recurso a estratégias compensatórias de movimento na SMSP. Estas modificações sugerem um controlo postural mais eficiente.

Expression des nétrines dans la rétine de souris adulte

Les nétrines sont une petite famille de protéines de guidage axonal qui peuvent attirer des axones ou en repousser d’autres lors du développement neuronal. Lors du développement de la rétine, les axones des cellules ganglionnaires de la rétine (CGR) sont attirés vers une source de nétrines au niveau du disque optique leur permettant de quitter la rétine et de former le nerf optique. Afin de pouvoir caractériser le rôle des nétrines dans le système visuel adulte, nous avons investigué l’expression des nétrines et leurs récepteurs dans la rétine de souris adulte. Alors qu’aucune expression de la nétrine-1 n’a été détectée dans la rétine adulte, l’expression de la nétrine-3 a été abondamment détectée au niveau des CGR et des cellules amacrines. Nous démontrons aussi que les récepteurs des nétrines sont exprimés dans la rétine adulte. Alors que DCC semble être confiné au niveau des axones des CGR, néogénine est retrouvé dans les dendrites des CGR et des cellules horizontales. Quant aux protéines de la famille des récepteurs homologues à UNC-5, UNC5B a été détecté dans les somas des CGR et UNC5C dans les cellules de Müller. La découverte que nétrine-3 et ses récepteurs sont abondamment exprimés dans plusieurs types cellulaires de la rétine adulte leur suggère un rôle dans le fonctionnement du système visuel mature.

Estrogens and thyroid hormone: Non-genomic effects are coupled to transcription.

Estrogens and thyroid hormones are regulators of important diverse physiological processes such as reproduction, thermogenesis, neural development, neural differentiation and cardiovascular functions. Both are ligands for receptors in the nuclear receptor superfamily, which act as ligand-dependent transcription factors, regulating transcription. However, estrogens and thyroid hormones also rapidly (within minutes or seconds) activate kinase cascades and calcium increases, presumably initiated at the cell membrane. We discuss the relevance of both modes of hormone action, including the membrane estrogen receptor, to physiology, with particular reference to lordosis behavior. We first showed that estrogen restricted to the membrane can, in fact, lead to subsequent increases in transcription from a consensus estrogen response element-based reporter in the neuroblastoma cell line, SK-N-BE(2)C. Using a novel hormonal paradigm, we also showed that the activation of protein kinase A, protein kinase C, mitogen activated protein kinase and increases in calcium were important in the ability of the membrane-limited estrogen to potentiate transcription. We discuss the source of calcium important in transcriptional potentiation. Since estrogens and thyroid hormones have common effects on neuroprotection, cognition and mood, we also hypothesized that crosstalk could occur between the rapid actions of thyroid hormones and the genomic actions of estrogens. In neural cells, we showed that triiodothyronine acting rapidly via MAPK can increase transcription by the nuclear estrogen receptor ERa from a consensus estrogen response element, possibly by the phosphorylation of the ERa. Novel mechanisms that link signals initiated by hormones from the membrane to the nucleus are physiologically relevant and can achieve neuroendocrine integration

Ontogenetic expression of the vanilloid receptors TRPV1 and TRPV2 in the rat retina

The present study aimed to analyze the gene and protein expression and the pattern of distribution of the vanilloid receptors TRPV1 and TRPV2 in the developing rat retina. During the early phases of development, TRPV1 was found mainly in the neuroblastic layer of the retina and in the pigmented epithelium. In the adult, TRPV1 was found in microglial cells, blood vessels, astrocytes and in neuronal structures, namely synaptic boutons of both retina] plexiform layers, as well as in cell bodies of the inner nuclear layer and the ganglion cell layer. The pattern of distribution of TRPV1 was mainly punctate, and there was higher TRPV1 labeling in the peripheral retina than in central regions. TRPV2 expression was quite distinct. its expression was virtually undetectable by immunoblotting before P1, and that receptor was found by immunohistochemistry only by postnatal day 15 (PI 5). RNA and protein analysis showed that the adult levels are only reached by P60, which includes small processes in the retinal plexiform layers, and labeled cellular bodies in the inner nuclear layer and the ganglion cell layer. There was no overlapping between the signal observed for both receptors. in conclusion, our results showed that the patterns of distribution of TRPV1 and TRPV2 are different during the development of the rat retina, suggesting that they have specific roles in both visual processing and in providing specific cues to neural development. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Investigação dos efeitos protetores do selenito de sódio sobre a neurotoxicidade do metilmercúrio em diferentes períodos de desenvolvimento de ratos wistar

A exposição a compostos mercuriais resulta em danos oxidativos, afetando gravemente o sistema nervoso central, como observado em humanos e em modelos experimentais. Este trabalho utilizou ratos Wistar em diferentes períodos do neuro-desenvolvimento a fim de investigar possíveis efeitos protetores do selênio (selenito de sódio) em um modelo in vivo de exposição ao metilmercúrio (MeHg). Os sujeitos (grupos de idades P1 e P21) receberam por amamentação ou via oral: veículo, Selênio (5ppm), MeHg (10ppm) ou Selênio (5ppm) mais MeHg (10ppm) durante 20 e 10 dias respectivamente (n = 8 por grupo). Após o tratamento, os ratos foram submetidos aos testes de campo aberto e labirinto aquático a fim de analisar déficits motores e de memória/aprendizagem, respectivamente. Para fins de análise histológica, foi realizada perfusão e imunohistoquimica para Neu-N. Com o objetivo de aferir possíveis efeitos deletérios sobre populações neuronais, foi feita contagem estereológica dos neurônios do hipocampo (camada polimórfica do giro denteado). Como resultado, foi observada redução significativa na atividade locomotora de neonatos (P1) mediante exposição ao MeHg. Além disso, nos grupos expostos ao MeHg (isoladamente ou associado ao selênio) verificou-se déficits de aprendizagem e memória. Já os animais P21 expostos ao MeHg apresentaram aumento na atividade locomotora, efeito abolido pela administração concomitante de selênio. Quando submetidos ao labirinto aquático, observou-se redução do tempo de latência apenas no grupo controle e naqueles animais expostos ao selênio. Como resultado das contagens estereológicas, observou-se diminuição do número de neurônios no hipocampo somente nos animais P21 expostos ao mercúrio. Os resultados obtidos sob estas condições experimentais mostraram que a exposição ao MeHg resultou em efeitos comportamentais diversos dependentes da idade dos sujeitos. A administração de selênio só foi capaz de interferir positivamente nos déficits locomotores observados em animais mais velhos. Além disso, foi observado que a administração de selênio não interferiu nos distúrbios comportamentais de memória/aprendizagem, tampouco na morte neuronal induzida por MeHg. Possíveis mecanismos associados a este padrão de proteção parcial por selênio, especialmente em estágios mais avançados de desenvolvimento neural ainda necessitam ser elucidados.