Дисертації з теми "EphB4 receptor"

EphB4, a known venous marker, represents a potential therapeutic target in modern vascular medicine. This study looked at the role of EphB4 as it pertains to basic cell functions in a mouse lung endothelium model (MLEC). Basic science techniques of microscopy, blotting and antibody labeling were used to evaluate and measure cellular response to EphB4 stimulation and manipulation. We found significant changes in MLEC cellular functions due to heterozygous knockout of the EphB4 receptor. These changes included decreased cellular migration and proliferation in knockout cells. We also saw increases in other cellular functions, such as tube formation and nitric oxide formation. From these data we were able to conclude that EphB4 is an active kinase in differentiated cells with a significant inhibitory effect. In EphB4 +/- knockout cell lines there was a lack of EphB4 inhibition and AKT and ERK showed increased activity. This work clearly implicates EphB4 as a major regulator of the basic cellular function of endothelia and highlights the need for further investigation into the specific pathways by which it functions.

This thesis presents insight into the signalling pathways of EphB4, a protein known to be involved in cancer progression. A new method was developed and used to identify a number of proteins that can interact with EphB4; and a novel mechanism of action for EphB4 was identified, where EphB4 was transported outside of the host cell by small vesicles called exosomes. These findings suggest that EphB4 may play important roles in pathways in both normal and disease states, and opens up new research directions into understanding the mechanisms, signalling pathways and cellular consequences of these new roles for EphB4.

EphB4 is a receptor protein over-expressed by many different cancers. This study explored the regulation of EphB4 and its binding partner, the ephrin-B2 ligand, in prostate cancer cells. This work showed that both EphB4 and ephrin-B2 can be cleaved by an important prostate cancer associated protease, KLK4 and this regulates the interaction between EphB4 and ephrin-B2 to activate different biological responses which could contribute to the initiation and progression of prostate cancer. This is a novel mechanism, that with further investigation, may provide new options for the development of anti-cancer therapies.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
A colonização do timo por precursores hematopoéticos representa um evento crucial para o desenvolvimento deste próprio órgão, assim como garante a diferenciação e a formação do repertório de células T maduras. Entretanto, os mecanismos moleculares que dirigem este processo não são totalmente conhecidos. A entrada destes precursores depende da ativação de uma cascata de sinalizações intermoleculares, onde participam algumas moléculas, como as integrinas e as quimiocinas. Os receptores Eph, que compõem a maior família de receptores tirosina-quinase, representam importantes moléculas reguladoras do desenvolvimento de sistemas e órgãos, sendo encontrados também no tecido linfóide. Mais recentemente, essa família de receptores, juntamente com seus ligantes, efrinas, foi descrita como moléculas co-estimulatórias de sinais transmitidos em linfócitos T pelo receptor de antígeno, por quimiocinas e integrinas. Neste contexto, o objetivo central deste trabalho foi o de avaliar as possíveis funções dos receptores Eph, em particular EphB2, em modular a atividade migratória de precursores T durante os processos de colonização do timo e maturação intratímica de linfócitos Nossos resultados demonstram a expressão dos receptores EphB2 no timo de camundongos e a sua participação tanto nos processos iniciais da organogênese do timo, quanto na diferenciação intratímica de timócitos. Este receptor, assim como seus principais ligantes, também é expresso em células precursoras derivadas da medula óssea de camundongos e é capaz de modular a migração e a capacidade de entrada destes precursores em lóbulos tímicos alinfóides. Além disso, vimos que a falta deste receptor, ou de seu domínio catalítico tirosina-quinase, promove uma redução na deposição de proteínas da matriz extracelular e de quimiocinas no timo, assim como resulta em importante inibição da entrada dos precursores hematopoiéticos neste órgão. De igual maneira, o desequilíbrio dos sinais transmitidos pelo complexo EphB2/efrina-B impede o correto posicionamento intratímico destes precursores, possivelmente levando a um bloqueio na maturação dos timócitos. Finalmente, demonstramos que a ausência do receptor ou dos sinais EphB2 não modifica os níveis de expressão de outros receptores como integrinas e receptores de quimiocina nos precursores hematopoiéticos e timócitos, mas possivelmente modula sua atividade e, desta forma, a atividade migratória destas células frente a estímulos hapto e quimiotáticos. Em conjunto, nossos resultados apontam uma importante participação dos sinais desencadeados pelo complexo Eph/efrina e sua co-regulação com outros receptores que modulam o processo de migração dos precursores de células T, desde sua entrada no timo, até o seu correto desenvolvimento e migração dentro deste órgão
Thymus settling by hematopoietic progenitors represents a crucial event during thymus ontogeny and guarantees the proper differentiation of the T-cell repertoire. However, the molecular mechanisms that drive such process are not completely understood. Progenitor settling depends on the activation of intercellular signaling cascades, where some integrins and chemokines play a role. Eph receptors, th e major tyrosine-kinase receptor family, are important regulatory molecules for the development of several systems and organs, being also expressed in lymphoid tissues. More recently, this receptor family, conjointly with the corresponding ligands, the ephrins, has been reported as costimulatory molecules for the T- cell receptor, chemokine receptors and integrins on T lymphocytes. In this context, the aim of this work was to evaluate the possible functions of Eph receptors, in particular EphB2, as modulators of T-cell progenitor migration during thymus settling and intrathymic T-cell maturation. Our results demonstrate that EphB2 receptors are expressed in the mouse thymus and participate in its organogenesis and intrathymic T-cell development. This receptor and its main ligands are also expressed in mouse bone marrow-derived progenitor cells, being able to modulate migration and the ability of these cells to settling thymic lobes. Moreover, the lack of such receptor, or its tyrosine-kinase domain, results in a reduced deposition of extracellular matrix proteins and chemokines in the thymus, and leads to an important inhibition of thymus settling by hematopoietic progenitors. Furthermore, an imbalance of the signals transmitted by EphB2/ephrin-B complex prevents proper intrathymic positioning of progenitor cells, possibly causing a blockade in thymocyte maturation. Finally, we demonstrated that the lack of EphB2 receptor or signaling does not change the expression level of integrins and chemokine receptors on hematopoietic progenitors and thymocytes, but possibly modulates the activity of these receptors and the cell migration activity through hapto and chemotactic stimuli. Taken together, our results point to an important participation of Eph/ephrin complex signaling and its cross-regulation with other receptors that modulates T-cell migration process, from thymus settling until the pr oper thymocyte development within the organ.

EphB receptor tyrosine kinases regulate cell-cell contacts throughout nervous system development, mediating processes as diverse as axon guidance, topographic mapping, neuronal migration and synapse formation. EphBs bind to a group of ligands, ephrin-Bs, which span the plasma membrane, thus allowing for bidirectional signaling between cells. Since EphBs are capable of multiple modes of signaling, and since they regulate numerous interdependent stages of development, it has been challenging to define which signaling functions of EphBs mediate particular developmental events. To overcome this hurdle, we developed an approach combining chemical biology with genetic engineering to reversibly inhibit EphB receptors in vivo. By mutating a residue in the receptor’s ATP-binding pocket, we rendered its kinase activity sensitive to reversible inhibition by PP1 analogs that do not inhibit wild type receptors. We engineered triple knockin mice bearing this mutation in which the kinase activity of EphB1, EphB2, and EphB3 can be rapidly, reversibly, and specifically blocked. Since we are able to block the kinase activity of EphBs while leaving their scaffolding and reverse signaling capabilities intact, we can precisely isolate the role of the kinase domain. In addition, acute inhibition can circumvent the developmental compensation that may occur after genetic mutations and can even allow the controlled study of EphBs in the mature brain and in disease models. Using these mice, termed analog-sensitive EphB triple knockin (AS-EphB TKI) mice, we demonstrate a requirement for the kinase-dependent signaling of EphBs in the collapse of retinal ganglion cell growth cones in vitro and the guidance of retinal axons at the optic chiasm in vivo. In addition, we show that the formation of several cortical axon tracts, including the corpus callosum, requires EphB tyrosine kinase signaling. In contrast, we find that steps in synapse development that are thought to be EphB-dependent occur normally when the kinase activity of EphBs is inhibited. We conclude that a cardinal in vivo function of EphB signaling, the ability to mediate axon guidance via growth cone repulsion, requires the tyrosine kinase activity of EphBs, while the development of functional excitatory synapses is independent of EphB tyrosine kinase activity.

Traumatic brain injury (TBI) can cause a number of deleterious effects to the neurovascular system, including reduced cerebral blood flow (CBF), vascular regression, and ischemia, resulting in cognitive decline. Research into therapeutic targets to restore neurovascular function following injury has identified endothelial EphA4 receptor tyrosine kinase as a major regulator of vascular regrowth. The research outlined herein utilizes an endothelial-specific EphA4 knockout mouse model (KO-EphA4flf/Tie2-Cre) to determine the extent to which this receptor may influence vascular regrowth following TBI. Analysis of the colocalization and proximity of endothelial and mural cell markers (i.e. PECAM-1 and PDGFRβ, respectively) in immunohistochemically-stained brain sections demonstrates that EphA4 silencing does not seem to affect the physical association between, nor total amounts of, endothelial cells and pericytes, between genotypes by 4 days post-injury (dpi). Nevertheless, these measures demonstrate that these cell types may preferentially proliferate and/or expand into peri-lesion tissue in both KO-EphA4flf/Tie2-Cre) and WT-EphA4fl/fl mice. These data further suggest that both genotypes experience homogeneity of PECAM-1 and PDGFRβ expression between regions of the injury cavity. Gene expression analysis using mRNA samples from both genotypes reveals that KO-EphA4flf/Tie2-Cre CCI-injured mice experience increased expression of Vegfa, Flt1, and Fn (Fibronectin) compared to sham-injured condition knockouts. These results demonstrate changes in expression of angiogenic factors in the absence of early differences in patterns of vessel formation, which may underlie improved vascular regrowth, as well as outline a potential mechanism wherein the interplay between these factors and EphA4 silencing may lead to improved cognitive outcomes following TBI.
Master of Science
Every day in the United States, an average of 155 people die due to the consequences of traumatic brain injury (TBI), with many survivors suffering life-long debilitating effects, including deficits in behavior, mobility, and cognitive ability. Because of this, there is a need for researchers to identify therapeutic strategies to stimulate recovery and improve patient outcomes. Recent advancements in the field of vascular biology have identified the regrowth of the blood vessels in the brain following TBI-induced damage as an important step in the recovery process, since the resulting increases in blood flow to damaged tissue will provide oxygen and nutrients necessary to fuel recovery. The work presented in this Masters thesis follows in this vein by examining a protein receptor known as EphA4, which is found on cells within blood vessels and has been implicated in reducing the rate of vessel growth under injury conditions. By blocking the activity of EphA4, we hoped to find increased vascular regrowth following brain injury in mice. During the experiments outlined herein, it was found that there were no statistically significant differences in vessel-associated cell densities between mice with or without EphA4 activity 4 days after injury, but there were differences in the levels of proteins and/or signals associated with vessel growth. Based on these results, we conclude that removing EphA4 activity increases expression of these pro-vessel growth proteins in mouse brains following injury at these early time points, potentially leading to increased vessel growth and improved recovery over subsequent weeks following injury.

In the intestinal epithelium, Wnt signaling drives the expression of the genes encoding tyrosine kinase receptors EphB2 and EphB3 and represses the expression of their membrane-tethered ligands, ephrin-Bs. Eph-ephrin interactions result in cellular repulsion and are involved in boundary formation. The project of this thesis is to understand the mechanism by which EphB−ephrin-B signals restrict cell positioning of cell types (cell sorting) in the normal intestinal epithelium and suppress colorectal cancer (CRC) progression beyond the earliest stages. We have demonstrated that at the onset of CRC EphB receptors impair the expansion of tumor cells through a mechanism dependent on E-cadherin–mediated adhesion. We show that EphB-mediated compartmentalization restricts the spreading of EphB+ tumor cells into ephrin-B1+ territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1+ intestinal cells at the onset of tumorigenesis. We have discovered that cell sorting is the outcome of two integrated mechanisms: cell contraction/repulsion and differential cell adhesion. The latter is the driving force to induce EphB/ephrin-B−mediated cell compartmentalization. We have developed in vitro models to analyze the mechanisms that induce E-cadherin remodeling upon EphB activation. We found RhoA, p120-catenin and the metalloproteinase ADAM10 as downstream effectors of EphB signaling involved in the control of cell sorting in CRC cells.
A l'epiteli intestinal, la ruta de senyalització Wnt indueix l'expressió dels gens que codifiquen per als receptors tirosina kinasa EphB2 i EphB3 i reprimeixen la dels seus lligands transmembrana, efrines de tipus B. Les interaccions Eph-efrina causen repulsió cel·lular i estan implicades en la formació de fronteres entre compartiments. La finalitat d'aquesta tesi és entendre el mecanisme pel qual la senyalització per EphB−efrina-B restringeix el posicionament dels diferents tipus cel·lulars a l'epiteli intestinal normal i suprimeix la progressió del càncer colorectal (CRC) en els primer estadis. Hem demostrat que, a l’inici del CRC, els receptors EphB restringeixen l'expansió de les cèl·lules tumorals a través d'un mecanisme depenent d'adhesió intercel·lular a través d’E-cadherina. En aquest treball es mostra in vitro i in vivo que la compartimentalització mitjançada per la senyalització dels receptors EphB restringeix l’invasió de les cèl·lules tumorals EphB+ als territoris efrina-B+. Aquests resultats indiquen que les cèl·lules de CRC han de silenciar l’expressió d'EphB per evitar les interaccions repulsives imposades per les cèl·lules intestinals normals efrina-B+ circumdants al començament del procés de tumorigènesi. Hem pogut discernir que el reordenament cel·lular per senyals EphB−efrina-B és el resultat de dos mecanismes integrats: la contracció/repulsió intercel·lular i l’adhesió diferencial entre diferents poblacions cel·lulars. Aquesta última és la força principal que condueix a la compartimentalització cel·lular mitjançada per EphB−efrina-B. Hem desenvolupat models in vitro per analitzar els mecanismes que provoquen el remodelament de la E-cadherina sota la senyalització per EphB. Presentem RhoA, p120-catenina i ADAM10 com a efectors de la senyalització de la ruta EphB implicats en el control de la compartimentalització cel·lular en el CRC.

Traumatic Brain Injury (TBI) mainly affects young persons in traffic accidents and the elderly in fall accidents. Improvements in the clinical management have significantly improved the outcome following TBI but survivors still suffer from depression, memory problems, personality changes, epilepsy and fatigue. The initial injury starts a series of events that give rise to a secondary injury process and despite several clinical trials there is no drug available for clinical use that targets secondary brain injury mechanisms. Some recovery of function is seen during the first months following injury but is usually limited and there are no drugs that stimulate the recovery of lost function. Some of the recovery is attributed to plasticity, the brains ability to adapt to new circumstances, and enhancing plasticity via increased axonal growth has the potential to partly restore lost function. In this thesis mice were subjected to the controlled cortical impact model of TBI and functional outcome was evaluated using Morris water maze, the cylinder test and the rotarod. Brain tissue loss was measured in all Papers but the additional histological analyses differ among the Papers. Attempts to increase axonal growth were made by interfering with Nogo receptor function in Paper I and by conditional knockout of ephA4 in Paper II. Contrary to the hypothesis cognition was impaired in Paper I but otherwise no effects of treatment were detected in Paper I and II. Much is still unknown about plasticity and despite the discouraging results of Papers I and II this treatment approach is still worth further exploration. It is firmly established that TBI results in an inflammatory response and some aspects of it may damage brain tissue. In Papers III and IV the inflammatory response was attenuated using an IL-1β directed antibody which resulted in reduced tissue loss and edema while improving cognitive function. The results from Papers III and IV are encouraging and the possibility to find a treatment based on IL-1β inhibition appears promising.

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.

La myosine 1b, un moteur moléculaire non-conventionnel, est impliquée dans une variété de processus cellulaires, contrôlant, par exemple la morphologie endomembranaire, le développement des axones et la ségrégation cellulaire. Le mécanisme par lequel la myosine 1b est capable de remplir ses fonctions dans une variété de régions cellulaires reste inconnu à ce jour, mais les phénotypes décrits suggèrent un rôle de la myosine 1b à l'interface entre les membranes et l'actine. Notamment, elle est nécessaire pour une ségrégation cellulaire efficace après l'activation du récepteur EphB2 qui induit la contraction cellulaire.Cette thèse présente une caractérisation détaillée des effets de la myosine 1b sur (1) les propriétés mécaniques de la membrane cellulaire, étudiées par tirage de tubes membranaires à l’aide d’une pince optique, et (2) la dynamique du cytosquelette d'actine et des protéines transmembranaires, étudiées à l’aide d’une variété de méthodes basées sur l’imagerie microscopique.Dans cette thèse nous montrons que les myosines de classe 1 ne changent pas généralement la tension membranaire effective dans les cellules adhérentes, probablement en raison de mécanismes de compensation efficaces. De plus, nous montrons que la friction entre le cortex d'actine et la membrane plasmique dépend de la densité totale des liens entre membrane et cortex et de la fraction relative des protéines liées. L’inefficacité de la contraction cellulaire observée en absence de la myosine 1b est donc indépendante d'un changement global et persistant de la tension membranaire effective.Dans la deuxième partie de cette thèse, nous montrons que la myosine 1b ne modifie pas la dynamique du récepteur EphB2, c'est-à-dire son comportement de diffusion et de clustering, dans la membrane plasmique.Enfin, en utilisant la microscopie TIRF-SIM et une description quantitative des flux d'actine, nous révélons que la myosine 1b a un effet intrigant mais non-intuitif sur la dynamique de l'actine à la surface ventrale des cellules.En conclusion, même si le mécanisme par lequel la myosine 1b change la réponse cellulaire après stimulation des récepteurs EphB2 reste encore inconnu, nous avons finalement été en mesure de lier sa fonction à une observation bien définie et quantifiable, à savoir la modification de la dynamique des flux d'actine. Les expériences futures seront en mesure de répondre à cette observation et de disséquer son mécanisme sous-jacent. Cela permettra de conclure si la myosine 1b a un effet commun qui régit tous ses rôles biologiques décrits
The unconventional motor protein myosin 1b is involved in a variety of cellular processes, controlling, e.g. endomembrane shape, axon development, and cell segregation. The mechanism by which myosin 1b is able to fulfil its functions in a variety of cellular locations remains unknown to date, yet the described phenotypes suggest a role of myosin 1b at the interface between membranes and actin. Notably, it is required for efficient cell segregation after activation of the EphB2 receptor which induces cell contraction.This thesis presents a detailed characterization of the effects of myosin 1b on (1) the mechanical properties of the cell membrane, studied by membrane tether pulling with an optical tweezer, and (2) the dynamics of the actin cytoskeleton and transmembrane proteins, studied by a variety of microscopy-based methods.Here we show that class 1 myosins do not generally change effective membrane tension in adherent cells, likely due to efficient compensation mechanisms. Furthermore, we show that friction between the actin cortex and the plasma membrane depends on the total density of membrane-cortex linkers and the relative fraction of bound proteins. The observed deficiency in cell contraction in absence of myosin 1b is thus independent of a persistent, global change in effective membrane tension.In the second part of this thesis, we show that myosin 1b likely does not change EphB2’s receptor dynamics in the plasma membrane, i.e. its diffusion and clustering behavior.Finally, using TIRF-SIM imaging and quantitative description of actin flows, we reveal that myosin 1b has an intriguing yet non-intuitive effect on actin dynamics at the cellular ventral surface.In conclusion, even if the mechanism by which myosin 1b changes cellular response to EphB2 stimulation still remains unknown, we have finally been able to pinpoint its function to a well-defined and quantifiable observation, i.e. changed actin flow dynamics. Future experiments will be able to address this observation and dissect its underlying mechanism. This will allow concluding on whether myosin 1b has a common effect that governs all its described biological roles

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.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in Australia and globally. Early detection and intervention is vital for the longevity of patients with any cancer, however, this appears to be most challenging with CRC, as it is largely asymptomatic. For this reason, most cases are not diagnosed until the cancer has metastasised, primarily to the liver. At this late stage of diagnosis, 5- year patient’s survival is predicted to be less than 10%. However, even when CRC is diagnosed and treated in the initial stages of neoplastic growth, high recurrence rates in patients still present as a serious issue. The problems associated with treatment and recurrence raise the need to identify molecular targets, so that specific and aggressive therapeutic interventions may be designed and developed. One such potential target is the erythropoietin-producing hepatocellular B4 (EphB4) receptor. The Ephs constitute the largest family of tyrosine kinase receptors. The activation of Eph receptors is achieved through association with their corresponding cell- bound ‘Eph receptor interacting’ (Ephrin) ligands. The signalling by the Eph receptors and their membrane-bound ligands, the Ephrins, is unique among the tyrosine kinases as both the receptor and ligand are found on the cell surface. Bidirectional interaction results in the phenomena of ‘forward’ signalling via the Eph receptor carrying cells and ‘reverse’ signalling in those cells expressing the Ephrin ligands. Several members of the Eph receptor receptor family, including EphB4, have been implicated with progression of many different types of cancer. However, EphB4 receptor’s contribution towards CRC yields the most contradictory findings. Some studies suggest that EphB4 is upregulated in late and metastatic stages of CRC, while others argue that EphB4 expression is often silenced in the progressive state of the disease. Due to the promising results achieved in other types of cancers, it is important to elucidate the role of EphB4 receptors in CRC in order to develop more specific and aggressive cancer therapies. The overall aim of this study is to elucidate the influence of EphB4 receptor expression on the development and progression of CRC. To achieve this, we used modified derivatives of multiple human and a mouse CRC cell line in in vitro and in vivo experiments. In vitro experiments were utilised to study effects of EphB4 overexpression and knockout on proliferative aptitude, migratory and invasive abilities of human and mouse CRC cells. In vivo subcutaneous models of CRC were used to evaluate the ability of high, low and knockdown of EphB4 receptor expression to influence morphological changes, rate of growth, vascularization and tumour-stromal interactions. The time course and rate of metastasis of CRC cells to the liver were studied in in vivo orthotopic and intra-splenic metastasis models. The level of EPHB4 and EPHRINB2 expression was investigated using databases to determine their correlation with survival and disease-free outcomes of CRC patients. The results of this study provide evidence that high EphB4 receptor expression significantly increases the rate of proliferation, migration and invasion of CRC cells in vitro, and enhances tumour growth in vivo due to enhanced vascularisation. Knockout of EphB4 expression reduces these effects. EphrinB2 appears to inhibit proliferation in cells overexpressing EphB4 and its expression correlates with poor patient outcome.

Recently we have begun to investigate a novel role of EphB receptors in opiate-dependant analgesia. EphB2-β-galactosidase knockins demonstrate that EphB2 is persistently expressed within a number of neural pathways involved in MOR-mediated nociception in vivo and that EphB2 colocalizes with markers of the MOR at the cellular level in the spinal cord and dorsal root ganglia. Despite demonstrating wild-type levels of sensory and motor activity, EphB2 null mice exhibit a significantly altered analgesic response to repeated (but not naive) opiate exposure compared to controls. Investigation of EphB2 null mice and wild type animals revealed no differences in MOR protein levels or affinity. Analysis of this opiate-mediated tolerance suggests that associative phenomena play a substantial role in mediating the analgesic effects observed, possibly due to defeciencies in CA1-mediated learning. Therefore, loss of EphB2 may diminish context-dependent learning and that such learning plays a substantial role in regulating morphine-dependent tolerance.

Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signaling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative variant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.

Neural function depends on precise wiring of axon during development. Previous studies have demonstrated that the erythropoietin producing hepatocellular carcinoma (Eph) family of tyrosine receptor kinases is crucial for the proper development of a number of neural circuits in the mammalian central nervous system (CNS). Mice lacking Eph receptors have been shown to exhibit deficits in pathways which include the thalamocortical, callosal, retinal and corticospinal tract. Due to the large number of Eph family members, the relative contribution of each receptor to axon pathfinding and neural function remains elusive. In this thesis, I have addressed the function of EphA4, EphB2 and EphB3 in the regulating the formation of interhemispheric projections within the forebrain and motor axon connections within the spinal cord using EphA4, EphB2 and EphB3and combinatorial null mice. To perform a detailed examination of the process of axon guidance regulated by these receptors within the forebrain, high resolution magnetic resonance imaging (MRI), immunofluorescence and in vivo stereotactic fluorescent labeling were performed. This work resulted in the development and validation of MRI-based analytic tools performed using EphB2 mutants which we have previously shown to exhibit specific morphologic defects in the anterior commissure (AC). Analysis of EphA4 null mice using high resolution MRI revealed for the first time that in addition to errors of midline crossing, loss of EphA4 activity results in a positional reorganization of the rostral AC. Results demonstrate that while EphB2 and A4 each regulate distinct aspects of guidance within ACpp, these receptors also operate cooperatively to control the guidance of axons in the pars anterior of the AC, a pathway not been previously implicated in Eph-mediated guidance. With respect to the spinal cord, mice deficient in EphB2 and EphA4 display prominent axon guidance errors in the medial subsets of the lateral motor column (LMCm); neurons which normally innervate ventral limb musculature. Finally, I have addressed the functional effect which Eph mutants exhibit with respect to motor behavior by examining a detailed set of motor coordination parameters

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.