Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration. Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo. The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA. The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface. The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed. In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.

Angiogenesis is a crucial process under both physiological and pathological conditions. Vascular endothelial growth factor (VEGF) A and its cognate receptor, vascular endothelial growth factor receptor 2 (VEGFR2) are key regulators of angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by de novo synthesis, and by both exocytic and endocytic trafficking. VEGF-binding to VEGFR2 induces phosphorylation of key tyrosine residues located in the cytosolic domain of the receptor, followed by clathrin-mediated endocytosis and signal transduction leading to vascular morphogenesis. Disabled protein 2 (Dab2) is a cytosolic, clathrin-adaptor protein that is known to regulate endocytosis of certain cell surface receptors. Studies of Dab2 function have revealed its role in the development of embryonic vasculature. However, the mechanism of Dab2 function, particularly in conjunction with endosomal VEGFR2, remains poorly understood. Our results show that Dab2 interacts with VEGFR2 and that upon VEGF stimulation the two proteins co-localize within Rab5-positive early endosomes. Knockdown of Dab2 reduces levels of VEGF-induced phosphorylation of VEGFR2 at residue Y1175. This is significant because phosphorylation of VEGFR2-Y1175 is crucial for pro-angiogenic signal transduction. Moreover, knockdown of Dab2 causes an increased trafficking of VEGFR2 to late endosomes (LE). Finally, this altered VEGFR2 trafficking following Dab2 knockdown has major functional consequences for endothelial cells, as they are unable to undergo morphogenesis into tube-like structures in an in vitro assay of angiogenesis. Collectively, our data show that Dab2 plays a crucial role in VEGFR2 trafficking in the endocytic system and this impacts receptor signaling and endothelial cell morphogenesis during angiogenesis.

Thrombospondin-1 (TSP-1) binding to its membrane receptor CD47 results in an inhibtion of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) and a decrease in intracellular cGMP levels. This causes physiologic effects such as vasoconstriction and a rise in blood pressure. The mechanism by which TSP-1 binds to CD47 at the membrane to decrease sGC activity is largely unknown. CD47 can physically associate with a number of binding partners, including α(v)β₃ and vascular endothelial growth factor receptor 2 (VEGFR2). Binding of a C-terminal fragment of TSP-1 called E3CaG1 to CD47 leads to a rise in intracellular calcium ([Ca²⁺](i)), which decreases sGC activity via a phosphorylation event. Binding of E3CaG1 is also known to disrupt the interaction between CD47 and VEGFR2, leading to a decrease in endothelial nitric-oxide synthase (eNOS) activity and cGMP levels through an Akt signaling pathway. However, it is not known whether other membrane proteins associated with CD47 are required for E3CaG1 binding and a subsequent [Ca²⁺](i) increase. Plasmon-waveguide resonance (PWR) spectroscopy was employed to elucidate the mechanism of TSP-1 inhibition of sGC activity through membrane complexes involving CD47. Using PWR, I found E3CaG1 can bind specifically to CD47 within native Jurkat membranes with picomolar and nanomolar dissociation constants (K(d)), suggesting multiple CD47 complexes are present. Among these complexes, CD47/VEGFR2 was found to bind E3CaG1 with a picomolar K(d)and CD47/α(v)β₃ was found to bind E3CaG1 with a nanomolar K(d). In addition, the presence of an anti-VEGFR2 antibody inhibited the E3CaG1-induced calcium response, which suggested CD47 in complex with VEGFR2 was responsible for TSP-1 reduction of sGC activity. I show that when both CD47 and VEGFR2 are returned to a HEK 293T cell line that does not contain these receptors, an increase in [Ca²⁺](i) upon E3CaG1 binding is restored. Interestingly, E3CaG1 was also found to bind to VEGFR2 in complex with the integrin α(v)β₃ on CD47-null cell lines and their derivations, causing a decrease in [Ca²⁺](i) levels. Therefore, the third type 2 repeat and C-terminal domains of TSP-1 can cause both increases and decreases in calcium based upon the availability of protein complexes to which it binds.

Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins. Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro. In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application. The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules. In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use. / <p>QC 20141203</p>

v anglickom jazyku Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Juliána Klimová Supervisor: Mgr. Pavel Bárta, Ph.D. Name of the work: In vitro saturation study of 99m Tc-HYNIC-ramucirumab on SKOV3 cell line. The passive immunotherapy is based on the use of already active immune system components (monoclonal antibodies), which play an important role in cancer cells elimination in the organism. The active immunotherapy tries to stimulate an active anticancer response via an appropriate form of an immunization. When monoclonal antibodies bind to cancer cells, those cells become a selected target for the following removal. The enhancement of the anti- cancer affect of monoclonal antibodies is possible due to the attachment of therapeutic agents like cytostatics, toxins and radionuclides. This presented master thesis is focused on the radiolabeling of the monoclonal antibody ramucirumab, which is directed against the vascular endothelial growth factor type 2 (VEGFR 2), which is often present in cells of some types of cancerous diseases. Within the experimental work, at first, there was a conjugation of chelating agent succinimidyl-6-hydrazino-nicotinamide (HYNIC) on the monoclonal antibody. After this step, radionuclide 99m...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18177号 / 医博第3897号 / 新制||医||1004(附属図書館) / 31035 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 中畑 龍俊, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’implication des protéines tyrosines phosphatases (PTPs) dans la régulation de la signalisation et la médiation des fonctions cellulaires a été bien établie dans les dernières années. Cependant, les mécanismes moléculaires par lesquels les PTPs régulent les processus fondamentaux tels que l’angiogenèse demeurent méconnus. Il a été rapporté que l’expression de la PTP DEP-1 (Density-enhanced phosphatase 1) augmente avec la densité cellulaire et corrèle avec la déphosphorylation du récepteur VEGFR2. Cette déphosphorylation contribue à l’inhibition de contact dans les cellules endothéliales à confluence et diminue l’activité du VEGFR2 en déphosphorylant spécifiquement ses résidus catalytiques Y1054/1059. De plus, la plupart des voies de signalisation en aval du VEGFR2 sont diminuées sauf la voie Src-Gab1-AKT. DEP-1 déphosphoryle la Y529 de Src et contribue à la promotion de la survie dans les cellules endothéliales. L’objectif de cette thèse est de mieux définir le rôle de DEP-1 dans la régulation de l’activité de Src et les réponses biologiques dans les cellules endothéliales. Nous avons identifié les résidus Y1311 et Y1320 dans la queue C-terminale de DEP-1 comme sites majeurs de phosphorylation en réponse au VEGF. La phosphorylation de ces résidus est requise pour l’activation de Src et médie le remodelage des jonctions cellules-cellules dépendantes de Src. Ce remodelage induit la perméabilité, l’invasion et la formation de capillaires en réponse au VEGF. Nos résultats démontrent que la phosphorylation de DEP-1 sur résidu tyrosine est requise pour diriger la spécificité de DEP-1 vers son substrat Src. Les travaux révèlent pour la première fois un rôle positif de DEP-1 sur l’induction du programme angiogénique des cellules endothéliales. En plus de la phosphorylation sur tyrosine, DEP-1 est constitutivement phosphorylé sur la thréonine 1318 situé à proximité de la Y1320 en C-terminal. Cette localisation de la T1318 suggère que ce résidu pourrait être impliqué dans la régulation de la Y1320. En effet, nous avons observé que la T1318 de DEP-1 est phosphorylée potentiellement par CK2, et que cette phosphorylation régule la phosphorylation de DEP-1 sur tyrosine et sa capacité de lier et d’activer Src. En accord avec ces résultats, nos travaux révèlent que la surexpression du mutant DEP-1 T1318A diminue le remodelage des jonctions cellules-cellules et par conséquent la perméabilité. Nos résultats suggèrent donc que la T1318 de DEP-1 constitue un nouveau mécanisme de contrôle de la phosphorylation sur tyrosine et que ceci résulte en l’activation de Src et l’induction des fonctions biologiques des cellules endothéliales en réponse au VEGF. Suite à ces travaux dans les cellules endothéliales qui démontrent un rôle positif de DEP-1 dans la médiation des réponses angiogéniques, nous avons voulu approfondir nos connaissances sur l’implication potentielle de DEP-1 dans les cellules cancéreuses où l’activité de Src est requise pour la progression tumorale. Malgré le rôle connu de DEP-1 comme suppresseur tumoral dans différents types de cancer, nous avons émis l’hypothèse que DEP-1 pourrait promouvoir les fonctions biologiques dépendantes de Src telles que la migration et l’invasion dans les cellules cancéreuses. Ainsi, nous avons observé que l’expression de DEP-1 est plus élevée dans les lignées basales de cancer du sein qui sont plus invasives comparativement aux lignées luminales peu invasives. Dans les lignées basales, DEP-1 active Src, médie la motilité cellulaire dépendante de Src et régule la localisation des protéines impliquées dans l’organisation du cytosquelette. L’analyse d’un micro-étalage de tissu a révélé que l’expression de DEP-1 est associée avec une réduction tendencielle de survie des patients. Nos résultats proposent donc, un rôle de promoteur tumoral pour DEP-1 dans la progression du cancer du sein. Les travaux présentés dans cette thèse démontrent pour la première fois que DEP-1 peut agir comme promoteur des réponses angiogéniques et du phénotype pro-invasif des lignées basales du cancer du sein probablement du à sa capacité d’activer Src. Nos résultats suggèrent ainsi que l’expression de DEP-1 pourrait contribuer à la progression tumorale et la formation de métastases. Ces découvertes laissent donc entrevoir que DEP-1 représente une nouvelle cible thérapeutique potentielle pour contrer l’angiogenèse et le développement du cancer. / The implication of protein tyrosine phosphatases (PTPs) in the regulation of cell signalling events and the mediation of cellular functions in response to growth factors such as VEGF has been well-established in the last years. Nonetheless, molecular mechanisms by which PTPs regulate fundamental processes such as angiogenesis are not well-characterized. Expression of the PTP DEP-1 (Density-enhanced phosphatase 1) was reported to increase with cell density and was associated with VEGFR2 dephosphorylation contributing to cell contact inhibition in confluent endothelial cells. We previously demonstrated that DEP-1 attenuates VEGFR2 activity by dephosphorylation of its Y1054/1059 leading to decreased activation of major signalling pathways downstream of VEGFR2 with exception of the Src-Gab1-AKT pathway. Increasing Src activity due to DEP-1-mediated dephosphorylation of its Y529 promotes endothelial cell survival. The objective of this thesis was to gain a better understanding of the role of DEP-1 in the regulation of the Src activity and of biological responses in endothelial cells. We identified tyrosine Y1311 and Y1320 in the C-terminal tail of DEP-1 as major phosphorylation sites in response to VEGF. These residues are required for Src activation and mediate the Src-dependent remodelling of endothelial cell junctions inducing permeability, invasion and capillary formation upon VEGF stimulation. We showed that VEGF-induced DEP-1 tyrosine phosphorylation directs DEP-1 specificity towards its substrate Src. Our results thus highlighted for the first time the promoting role of DEP-1 on the angiogenic program in endothelial cells. In addition to tyrosine phosphorylation, DEP-1 is constitutively phosphorylated on a threonine residue (T1318) proximal to Y1320 in its C-terminal tail suggesting it might be involved in the regulation of Y1320. Indeed, we found that DEP-1 T1318 is phosphorylated, potentially by CK2, and regulates the tyrosine phosphorylation of DEP-1 and its ability to bind to and activate Src. Consistent with this, remodelling of endothelial cell junctions and permeability are impaired in endothelial cells expressing the DEP-1 T1318 mutant. Thus, DEP-1 phosphorylation on T1318 displays a regulatory control over DEP-1 tyrosine phosphorylation and subsequently Src activation and endothelial cell functions in response to VEGF. Our results demonstrating that DEP-1 promotes angiogenic cell responses in endothelial cells, prompted us to consider a possible involvement of DEP-1 in cancer cells, where Src activation has been linked to cancer progression. Thus, although, DEP-1 is believed to act as a tumour suppressor in different cancer types, we hypothesized that it might also promote Src-dependent functions such as migration and invasion in cancer cells. Interestingly, we found that DEP-1 is higher expressed in more invasive basal-like breast cancer cells than in luminal-like cell lines. Moreover, DEP-1 is implicated in the regulation of Src activity, Src-mediated cell motility and appropriate localization of proteins mediating cytoskeletal organization in basal-like breast cancer cell lines. To further support these results, analysis of a breast cancer tissue microarray revealed that DEP-1 expression is associated with a tendency towards reduced overall survival. Thus, our results provide first evidence for a tumour-promoting role of DEP-1 in breast cancer. Altogether, the work performed in the context of this thesis revealed that DEP-1 can similarly behave as a promoter of the angiogenic response and of the pro-invasive phenotype in basal-like breast cancer cell lines, most likely due to its ability to activate Src. This suggests for the first time that DEP-1 expression could contribute to tumour progression and the formation of metastases, and as such, represent a potential new target for anti-angiogenic and anti-cancer therapy.

L’implication des protéines tyrosines phosphatases (PTPs) dans la régulation de la signalisation et la médiation des fonctions cellulaires a été bien établie dans les dernières années. Cependant, les mécanismes moléculaires par lesquels les PTPs régulent les processus fondamentaux tels que l’angiogenèse demeurent méconnus. Il a été rapporté que l’expression de la PTP DEP-1 (Density-enhanced phosphatase 1) augmente avec la densité cellulaire et corrèle avec la déphosphorylation du récepteur VEGFR2. Cette déphosphorylation contribue à l’inhibition de contact dans les cellules endothéliales à confluence et diminue l’activité du VEGFR2 en déphosphorylant spécifiquement ses résidus catalytiques Y1054/1059. De plus, la plupart des voies de signalisation en aval du VEGFR2 sont diminuées sauf la voie Src-Gab1-AKT. DEP-1 déphosphoryle la Y529 de Src et contribue à la promotion de la survie dans les cellules endothéliales. L’objectif de cette thèse est de mieux définir le rôle de DEP-1 dans la régulation de l’activité de Src et les réponses biologiques dans les cellules endothéliales. Nous avons identifié les résidus Y1311 et Y1320 dans la queue C-terminale de DEP-1 comme sites majeurs de phosphorylation en réponse au VEGF. La phosphorylation de ces résidus est requise pour l’activation de Src et médie le remodelage des jonctions cellules-cellules dépendantes de Src. Ce remodelage induit la perméabilité, l’invasion et la formation de capillaires en réponse au VEGF. Nos résultats démontrent que la phosphorylation de DEP-1 sur résidu tyrosine est requise pour diriger la spécificité de DEP-1 vers son substrat Src. Les travaux révèlent pour la première fois un rôle positif de DEP-1 sur l’induction du programme angiogénique des cellules endothéliales. En plus de la phosphorylation sur tyrosine, DEP-1 est constitutivement phosphorylé sur la thréonine 1318 situé à proximité de la Y1320 en C-terminal. Cette localisation de la T1318 suggère que ce résidu pourrait être impliqué dans la régulation de la Y1320. En effet, nous avons observé que la T1318 de DEP-1 est phosphorylée potentiellement par CK2, et que cette phosphorylation régule la phosphorylation de DEP-1 sur tyrosine et sa capacité de lier et d’activer Src. En accord avec ces résultats, nos travaux révèlent que la surexpression du mutant DEP-1 T1318A diminue le remodelage des jonctions cellules-cellules et par conséquent la perméabilité. Nos résultats suggèrent donc que la T1318 de DEP-1 constitue un nouveau mécanisme de contrôle de la phosphorylation sur tyrosine et que ceci résulte en l’activation de Src et l’induction des fonctions biologiques des cellules endothéliales en réponse au VEGF. Suite à ces travaux dans les cellules endothéliales qui démontrent un rôle positif de DEP-1 dans la médiation des réponses angiogéniques, nous avons voulu approfondir nos connaissances sur l’implication potentielle de DEP-1 dans les cellules cancéreuses où l’activité de Src est requise pour la progression tumorale. Malgré le rôle connu de DEP-1 comme suppresseur tumoral dans différents types de cancer, nous avons émis l’hypothèse que DEP-1 pourrait promouvoir les fonctions biologiques dépendantes de Src telles que la migration et l’invasion dans les cellules cancéreuses. Ainsi, nous avons observé que l’expression de DEP-1 est plus élevée dans les lignées basales de cancer du sein qui sont plus invasives comparativement aux lignées luminales peu invasives. Dans les lignées basales, DEP-1 active Src, médie la motilité cellulaire dépendante de Src et régule la localisation des protéines impliquées dans l’organisation du cytosquelette. L’analyse d’un micro-étalage de tissu a révélé que l’expression de DEP-1 est associée avec une réduction tendencielle de survie des patients. Nos résultats proposent donc, un rôle de promoteur tumoral pour DEP-1 dans la progression du cancer du sein. Les travaux présentés dans cette thèse démontrent pour la première fois que DEP-1 peut agir comme promoteur des réponses angiogéniques et du phénotype pro-invasif des lignées basales du cancer du sein probablement du à sa capacité d’activer Src. Nos résultats suggèrent ainsi que l’expression de DEP-1 pourrait contribuer à la progression tumorale et la formation de métastases. Ces découvertes laissent donc entrevoir que DEP-1 représente une nouvelle cible thérapeutique potentielle pour contrer l’angiogenèse et le développement du cancer. / The implication of protein tyrosine phosphatases (PTPs) in the regulation of cell signalling events and the mediation of cellular functions in response to growth factors such as VEGF has been well-established in the last years. Nonetheless, molecular mechanisms by which PTPs regulate fundamental processes such as angiogenesis are not well-characterized. Expression of the PTP DEP-1 (Density-enhanced phosphatase 1) was reported to increase with cell density and was associated with VEGFR2 dephosphorylation contributing to cell contact inhibition in confluent endothelial cells. We previously demonstrated that DEP-1 attenuates VEGFR2 activity by dephosphorylation of its Y1054/1059 leading to decreased activation of major signalling pathways downstream of VEGFR2 with exception of the Src-Gab1-AKT pathway. Increasing Src activity due to DEP-1-mediated dephosphorylation of its Y529 promotes endothelial cell survival. The objective of this thesis was to gain a better understanding of the role of DEP-1 in the regulation of the Src activity and of biological responses in endothelial cells. We identified tyrosine Y1311 and Y1320 in the C-terminal tail of DEP-1 as major phosphorylation sites in response to VEGF. These residues are required for Src activation and mediate the Src-dependent remodelling of endothelial cell junctions inducing permeability, invasion and capillary formation upon VEGF stimulation. We showed that VEGF-induced DEP-1 tyrosine phosphorylation directs DEP-1 specificity towards its substrate Src. Our results thus highlighted for the first time the promoting role of DEP-1 on the angiogenic program in endothelial cells. In addition to tyrosine phosphorylation, DEP-1 is constitutively phosphorylated on a threonine residue (T1318) proximal to Y1320 in its C-terminal tail suggesting it might be involved in the regulation of Y1320. Indeed, we found that DEP-1 T1318 is phosphorylated, potentially by CK2, and regulates the tyrosine phosphorylation of DEP-1 and its ability to bind to and activate Src. Consistent with this, remodelling of endothelial cell junctions and permeability are impaired in endothelial cells expressing the DEP-1 T1318 mutant. Thus, DEP-1 phosphorylation on T1318 displays a regulatory control over DEP-1 tyrosine phosphorylation and subsequently Src activation and endothelial cell functions in response to VEGF. Our results demonstrating that DEP-1 promotes angiogenic cell responses in endothelial cells, prompted us to consider a possible involvement of DEP-1 in cancer cells, where Src activation has been linked to cancer progression. Thus, although, DEP-1 is believed to act as a tumour suppressor in different cancer types, we hypothesized that it might also promote Src-dependent functions such as migration and invasion in cancer cells. Interestingly, we found that DEP-1 is higher expressed in more invasive basal-like breast cancer cells than in luminal-like cell lines. Moreover, DEP-1 is implicated in the regulation of Src activity, Src-mediated cell motility and appropriate localization of proteins mediating cytoskeletal organization in basal-like breast cancer cell lines. To further support these results, analysis of a breast cancer tissue microarray revealed that DEP-1 expression is associated with a tendency towards reduced overall survival. Thus, our results provide first evidence for a tumour-promoting role of DEP-1 in breast cancer. Altogether, the work performed in the context of this thesis revealed that DEP-1 can similarly behave as a promoter of the angiogenic response and of the pro-invasive phenotype in basal-like breast cancer cell lines, most likely due to its ability to activate Src. This suggests for the first time that DEP-1 expression could contribute to tumour progression and the formation of metastases, and as such, represent a potential new target for anti-angiogenic and anti-cancer therapy.

Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins. This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures. With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells. In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor. Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient. / (Faculty of Medicine)

Vandetanib, lenvatinib and cabozantinib are inhibitors of receptor tyrosine kinases approved to treat locally advanced or metastatic thyroid gland, kidney and liver cancers. These multi- kinase inhibitors, inhibit phosphorylation of tyrosine moieties of protein, thus modulate cell signalization in cancer cells. Metabolites of vandetanib, lenvatinib and cabozantinib were detected in vitro as well as in vivo in blood and urine. Cytochromes P450 and flavin monooxygenases were identified as primary enzymes participating in metabolism of these drugs. Literature lacks information regarding pharmacological efficacy of vandetanib, lenvatinib and cabozantinib metabolites. The aim of this diploma thesis was the investigation of pharmacological efficacy of N-oxides of vandetanib, lenvatinib and cabozantinib. The viability measurement under normoxic and hypoxic conditions was employed to determined their efficacy. The expression of enzymes of the first phase of xenobiotics metabolism (CYP 450 1A1, 1B1, 3A4 a CYP 450 oxidoreductase) and receptor tyrosine kinases RET and VEGFR2, as well as mechanism of changes in their expression were investigated using western blotting and flow cytometry. High performance liquid chromatography was utilised to investigate possible metabolism of tyrosine kinase inhibitors and...