Survival of transplanted neural stem/progenitor cells (NSPC) is limited after spinal cord injury (SCI). This thesis tested whether ephrin-B3 could enhance the survival of spinal cord derived NSPC because ephrin-B3 enhanced the survival of endogenous NSPC in the mouse brain. Preclustered ephrin-B3-Fc was tested, and preclustered Fc fragments and phosphate-buffered saline (PBS) were used as controls. This study showed that spinal cord derived NSPC and normal and injured rat spinal cord expressed EphA4 receptors. In culture, ephrin-B3-Fc increased the survival of NSPC at 1µg/mL (p<0.05), but Fc fragments reduced NSPC survival dose-dependently. In the injured spinal cord, infusion of ephrin-B3-Fc increased the proliferation of endogenous ependymal cells compared with infusion of PBS (p<0.05). However, in the injured cord, infusion of either ephrin-B3-Fc or Fc fragments caused a 20-fold reduction in the survival of transplanted NSPC (p<0.001). Thus, after SCI, ephrin-B3-Fc and Fc fragments are toxic to transplanted NSPC.

Survival of transplanted neural stem/progenitor cells (NSPC) is limited after spinal cord injury (SCI). This thesis tested whether ephrin-B3 could enhance the survival of spinal cord derived NSPC because ephrin-B3 enhanced the survival of endogenous NSPC in the mouse brain. Preclustered ephrin-B3-Fc was tested, and preclustered Fc fragments and phosphate-buffered saline (PBS) were used as controls. This study showed that spinal cord derived NSPC and normal and injured rat spinal cord expressed EphA4 receptors. In culture, ephrin-B3-Fc increased the survival of NSPC at 1µg/mL (p<0.05), but Fc fragments reduced NSPC survival dose-dependently. In the injured spinal cord, infusion of ephrin-B3-Fc increased the proliferation of endogenous ependymal cells compared with infusion of PBS (p<0.05). However, in the injured cord, infusion of either ephrin-B3-Fc or Fc fragments caused a 20-fold reduction in the survival of transplanted NSPC (p<0.001). Thus, after SCI, ephrin-B3-Fc and Fc fragments are toxic to transplanted NSPC.

The role that signaling molecules play during morphogenesis and their interactions is a field of intense study and the sea urchin represents a facile system to study these aspects of development in the early embryo. In many instances, the S. purpuratus genome contains relatively simple receptor-ligand signaling systems compared to vertebrate counterparts and this provides interesting opportunities to study their diversity of function during the morphogenetic events that shape the embryo. The Eph-Ephrin signaling components are an excellent example of this and they are represented by dozens of members in the vertebrate system with developmental functions that include axon guidance, cell migration and tissue segregation. In contrast, the sea urchin genome contains a single Eph receptor and a single Ephrin ligand and by interacting with different effectors of signal transduction, this simple, bipartite system can fulfill a variety of functional roles during morphogenesis. Studying the function of Eph-Ephrin signaling in the sea urchin embryo, I have revealed two distinct morphogenetic movements in which Eph-Ephrin signaling is necessary; apical constriction of ciliary band cells and pigment cell migration. In both examples, a functionally relevant Ephrin gradient establishes spatial information in the developing tissues, producing a reaction from cells expressing the Eph receptor. In the case of pigment cells, the distribution of migrating cells is affected and in the case of ciliary band cells, apical constriction occurs. The different outcomes of Eph-Ephrin signaling in these two tissues exemplifies signaling components communicating spatial information and initiating morphogenetic programs with outcomes dependent on cellular context. Furthermore, I have identified downstream components of Eph-Ephrin signaling that have necessary functions in both models, illustrating how different cellular programs can be induced by the same signaling iii iv components. My research contributes to understanding fundamental aspects of how complex 3 dimensional tissues arise from the genes and regulatory elements encoded in metazoan genomes. / Graduate

Ephrin type-B receptor 2 (EphB2) is a receptor tyrosine kinase which phosphorylates proteins and thereby regulates cell migration, vascular development, axon guidance synaptic plasticity, and formation of borders between tissues. It has been seen overexpressed in several cancers, which make it an interesting protein to study. In this thesis EphB2 kinase domain (KD) and juxtamembrane segment with kinase domain (JMS-KD) have been expressed, purified and studied using analytical ultracentrifugation to evaluate the oligomerisation of the KD and how the double mutation S677/680A affects this. A program for data analysis have been written and used for analysis of the acquired data. The values of the dissociation constant were 2.94±1.04 mM for KD wild type and 3.46±2.26 mM for JMS-KD wild type have been calculated. Due to varied problems with the measurements no data was acquired on the double mutant, and not enough data was gained to draw any conclusions. Additional experiments will be needed to understand the oligomerisation of this intriguing protein.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19249号 / 生博第342号 / 新制||生||46(附属図書館) / 32248 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 根岸 学, 教授 渡邊 直樹, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

EPHB2, an ephrin receptor (EPH) from receptor tyrosine kinase (RTK) family, is one of the substrates for tissue factor (TF) - coagulation factor VIIa (FVIIa) complex and it is cleaved in its ectodomain. EPHB2 cleavage is important for ephrin receptor (EPH) - ephrin ligand (EFN) signaling and cell repulsion. TF has been reported to be overexpressed in different cancer types such as breast and colorectal cancer (CRC). Furthermore, EPHB2 R155C mutation, at the TF/FVIIa-mediated cleavage site, has been identified as one of the somatic mutation sites in human metastatic CRC. Therefore, the aim of the present work was to characterize the EPHB2 R155C mutation and its effect on the cleavage by TF/FVIIa on EPHB2 in context to CRC. We generated overexpression cell models for EPHB2 wild type (wt) and R155C mutant in human CRC DLD-1 cell line for in vitro compartmentalization assay analysis to demonstrate repulsion event in EPH-EFN signaling. Whereas low endogenous TF expression led to incomplete cleavage of EPHB2 wt protein, stable overexpression of TF resulted in complete cleavage. Moreover, overexpression of TF resulted in reduced compartmentalization in EPHB2 wt cells after FVIIa treatment. Transient expression of TF in EPHB2 wt and R155C cells showed no clear difference in EPHB2 cleavage. Interestingly, it was difficult to obtain similar stable overexpression level of TF in EPHB2 R155C cells compared to EPHB2 wt cells. This may lead to further research in context to the role of TF/FVIIa-mediated EPHB2 cleavage in CRC by the generation of TF overexpression cell lines using lentiviral transduction.

No description available.

O fator derivado do epitélio pigmentado (PEDF) é um fator neurotrófico que possui um grande potencial trófico nos neurônios motores da medula espinal, bem como é capaz de modular o microambiente da lesão. Desta forma, analisamos a capacidade do tratamento com PEDF em promover a neuroplasticidade da medula espinal após lesão isquêmica. Ratos Wistar adultos foram submetidos à lesão medular isquêmica do tipo fototrombótica, segundo o método de Rose Bengal, na altura do 11° segmento torácico e foram imediatamente tratados com inoculação local de PEDF (grupo PEDF) ou solvente (grupo Salina). Ratos submetidos à cirurgia simulada (grupo Sham) receberam a injeção do solvente. Ao término do procedimento cirúrgico, os ratos foram submetidos a testes neurofuncionais durante 6 semanas. Após esse período, os animais sofreram eutanásia e o tecido medular foi dividido entre as técnicas de imunoistoquímica, western blot e PCR em tempo real. Foi analisada na região lombar anterior da medula espinal a modulação das CSPGs, a expressão dos fatores neurotróficos NT-3, GDNF, BDNF e FGF-2, bem como os níveis das moléculas associadas à angiogênese e apoptose (laminina e Bcl-2), das proteínas relacionadas à neuroplasticidade (MAP-2, GAP-43 e sinaptofisina) e do sistema Eph/efrina e a RhoA, que são capazes de modular o crescimento de fibras. Os resultados mostraram uma recuperação parcial e espontânea do comportamento sensório-motor dos animais que foram submetidos à lesão fototrombótica, onde o tratamento com PEDF foi capaz de potencializar alguns desses parâmetros. A análise da região lombar anterior da medula espinal, caudal à lesão, mostrou uma diminuição das CSPGs nos dois grupos lesados, o que pode ter favorecido os eventos de neuroplasticidade. O tratamento com PEDF foi capaz de promover a regulação dos fatores neurotróficos NT-3 e GDNF, diminuir a angiogênese local (diminuição da laminina) e potencializar o processo de neuroplasticidade (aumento da MAP-2) nessa região. A lesão medular isquêmica foi capaz de modular a expressão do receptor EphA4 e da efrina-B1 e o tratamento com PEDF possivelmente regulou o estado de ativação da efrina-A2 e da efrina-B3 e certamente modulou a ativação da efrina-B2. Ainda, os receptores Eph e as efrinas foram observados diferentemente nos neurônios e astrócitos. Nossos resultados confirmam a capacidade plástica da medula espinal após lesão e mostra que o tratamento com PEDF foi capaz de potencializar esse processo / The pigment epithelium derived factor (PEDF) is a neurotrophic factor that has a great trophic potential in the motor neurons of the spinal cord, and is able to modulate the lesion microenvironment. We analyzed the capacity of the treatment with PEDF to promote the neuroplasticity after ischemic spinal cord injury. Adult male Wistar rats were underwent to photothrombotic ischemic spinal cord injury, according to the Rose Bengal method, at the level of 11° thoracic segment, and were immediately treated with local injection of PEDF (PEDF group) or solvent (Saline group). Rats underwent to a sham surgery (Sham group) received solvent injection. At the end of surgery, the rats were submitted to neurofunctional tests during 6 weeks. After this period, the animals were euthanized, and the anterior lumbar region of the spinal cord tissue was submitted to immunohistochemistry, western blot and real-time PCR analyses. The inhibitory response of CSPGs, the expression of neurotrophic factors (NT-3, GDNF, BDNF and FGF-2), the molecules associated with angiogenisis and apoptosis (laminin and Bcl-2), the proteins related to neuroplasticity (MAP-2, GAP-43 and synaptophysin), as well the Eph/ephrin system and the RhoA, which is able to modulate the fibers growth, were evaluated. The results showed a spontaneous, and parcial, recovery of the sensory motor behavior of the animals that were underwent to a photothrombotic injury, and the treatment with PEDF was able to potentiate some of these parameters. The analysis of the anterior lumbar region of the spinal cord, caudally to the lesion, showed a decrease of CSPGs, which may have favored the neuroplasticity events. The treatment with PEDF was able to promote the regulation of NT-3 and GDNF, as well the reduction of laminin and the increase of MAP-2 in that region. In relation to the Eph/ephrin system, the ischemic spinal cord injury was able to modulate the EphA4 receptor and ephrin-B1 expression, and the treatment with PEDF possibly regulated the activation state of ephrin-A2 and ephrin-B3 and certainly modulated the ephrin-B2 activation. Eph receptors and ephrins have been found specifically in neurons and astrocytes. Our results confirmed the plastic capacity of the spinal cord after injury and showed that the treatment with PEDF was able to enhance this process

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

O fator derivado do epitélio pigmentado (PEDF) é um fator neurotrófico que possui um grande potencial trófico nos neurônios motores da medula espinal, bem como é capaz de modular o microambiente da lesão. Desta forma, analisamos a capacidade do tratamento com PEDF em promover a neuroplasticidade da medula espinal após lesão isquêmica. Ratos Wistar adultos foram submetidos à lesão medular isquêmica do tipo fototrombótica, segundo o método de Rose Bengal, na altura do 11° segmento torácico e foram imediatamente tratados com inoculação local de PEDF (grupo PEDF) ou solvente (grupo Salina). Ratos submetidos à cirurgia simulada (grupo Sham) receberam a injeção do solvente. Ao término do procedimento cirúrgico, os ratos foram submetidos a testes neurofuncionais durante 6 semanas. Após esse período, os animais sofreram eutanásia e o tecido medular foi dividido entre as técnicas de imunoistoquímica, western blot e PCR em tempo real. Foi analisada na região lombar anterior da medula espinal a modulação das CSPGs, a expressão dos fatores neurotróficos NT-3, GDNF, BDNF e FGF-2, bem como os níveis das moléculas associadas à angiogênese e apoptose (laminina e Bcl-2), das proteínas relacionadas à neuroplasticidade (MAP-2, GAP-43 e sinaptofisina) e do sistema Eph/efrina e a RhoA, que são capazes de modular o crescimento de fibras. Os resultados mostraram uma recuperação parcial e espontânea do comportamento sensório-motor dos animais que foram submetidos à lesão fototrombótica, onde o tratamento com PEDF foi capaz de potencializar alguns desses parâmetros. A análise da região lombar anterior da medula espinal, caudal à lesão, mostrou uma diminuição das CSPGs nos dois grupos lesados, o que pode ter favorecido os eventos de neuroplasticidade. O tratamento com PEDF foi capaz de promover a regulação dos fatores neurotróficos NT-3 e GDNF, diminuir a angiogênese local (diminuição da laminina) e potencializar o processo de neuroplasticidade (aumento da MAP-2) nessa região. A lesão medular isquêmica foi capaz de modular a expressão do receptor EphA4 e da efrina-B1 e o tratamento com PEDF possivelmente regulou o estado de ativação da efrina-A2 e da efrina-B3 e certamente modulou a ativação da efrina-B2. Ainda, os receptores Eph e as efrinas foram observados diferentemente nos neurônios e astrócitos. Nossos resultados confirmam a capacidade plástica da medula espinal após lesão e mostra que o tratamento com PEDF foi capaz de potencializar esse processo / The pigment epithelium derived factor (PEDF) is a neurotrophic factor that has a great trophic potential in the motor neurons of the spinal cord, and is able to modulate the lesion microenvironment. We analyzed the capacity of the treatment with PEDF to promote the neuroplasticity after ischemic spinal cord injury. Adult male Wistar rats were underwent to photothrombotic ischemic spinal cord injury, according to the Rose Bengal method, at the level of 11° thoracic segment, and were immediately treated with local injection of PEDF (PEDF group) or solvent (Saline group). Rats underwent to a sham surgery (Sham group) received solvent injection. At the end of surgery, the rats were submitted to neurofunctional tests during 6 weeks. After this period, the animals were euthanized, and the anterior lumbar region of the spinal cord tissue was submitted to immunohistochemistry, western blot and real-time PCR analyses. The inhibitory response of CSPGs, the expression of neurotrophic factors (NT-3, GDNF, BDNF and FGF-2), the molecules associated with angiogenisis and apoptosis (laminin and Bcl-2), the proteins related to neuroplasticity (MAP-2, GAP-43 and synaptophysin), as well the Eph/ephrin system and the RhoA, which is able to modulate the fibers growth, were evaluated. The results showed a spontaneous, and parcial, recovery of the sensory motor behavior of the animals that were underwent to a photothrombotic injury, and the treatment with PEDF was able to potentiate some of these parameters. The analysis of the anterior lumbar region of the spinal cord, caudally to the lesion, showed a decrease of CSPGs, which may have favored the neuroplasticity events. The treatment with PEDF was able to promote the regulation of NT-3 and GDNF, as well the reduction of laminin and the increase of MAP-2 in that region. In relation to the Eph/ephrin system, the ischemic spinal cord injury was able to modulate the EphA4 receptor and ephrin-B1 expression, and the treatment with PEDF possibly regulated the activation state of ephrin-A2 and ephrin-B3 and certainly modulated the ephrin-B2 activation. Eph receptors and ephrins have been found specifically in neurons and astrocytes. Our results confirmed the plastic capacity of the spinal cord after injury and showed that the treatment with PEDF was able to enhance this process