The pathogenesis of diabetic nephropathy (DN) remains incompletely understood. In previous studies, we observed the activation of Tyr kinase Src by high glucose (HG) and showed that Src is required for MAPK activation and synthesis of collagen IV in cultured rat mesangial cells (MCs). Reactive oxygen species (ROS) are also important mediators of DN, and our present study aimed to investigate the role of Src in HG-induced ROS generation. In MCs, we found that HG led to ROS accumulation that was blocked by Src inhibitors or Src-specific siRNA. Downstream of Src, Vav2 was phosphorylated/activated leading to Rac1-dependent NADPH oxidase activation. Long-term HG exposure resulted in Src-dependent Nox4 protein induction. Nox2-specific siRNA abrogated ROS production only in short-term HG, while Nox4-specific siRNA blocked ROS production only in long-term HG. Taken together, our data indicate Src to be important in mediating ROS generation from both Nox2- and Nox4-containing NADPH oxidases.

No description available.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains iv, 171 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 96-111).

Traditionally, drug discovery programs have focused on prioritising compounds by their affinity to a specific target in isolation, which was hypothesised to be the cause of a particular disease. Through chemical inhibition, the disease could, thus, be prevented or at the very least, controlled. These hypotheses require significant validation before drug screening can begin which relates to lengthy and expensive programs. Furthermore, drug screening against a single target in isolation is not a realistic model of cellular behaviour and is not appropriately tailored to more complex diseases such as cancer. Phenotypic drug discovery, on the other hand, bypasses any involvement of known targets, instead focusing on the desired outcome – the phenotype. In this way, drugs are biased by their potency on the phenotype and not against any particular targets. The molecular mechanism of action need not be known at all, however, it can be useful to later reveal the target(s) involved by various deconvolution methods. This thesis describes a cooperative ligand based phenotypic drug discovery approach, undertaken in order to develop more suitable small molecule drugs for cancer treatment. For this purpose, the promiscuous pyrazolopyrimidine inhibitor PP1 was chosen as a starting model compound. Modification of PP1 on the N1 position allowed a series of water solubilising groups to be incorporated into the pyrazolopyrimidine scaffold which created an initial 12-membered library. Testing against MCF7 breast cancer cells and looking at phenotypic end points such as cell proliferation, cell mobility and cell cycle, generated early target-agnostic structure/anti-proliferative activity relationships. These early results, along with compounds published in recent literature, were used to generate further libraries. Profiling lead compounds against a selection of 18 kinases known to be targeted by PP1, showed the compounds were inhibiting either SRC family or mTOR kinases which enabled the creation of two, structure specific, groups of inhibitors. Further lead optimisation led to the rapid discovery of preclinical candidates with excellent drug-like properties and potencies in both cellular assays and against their respective targets. Compounds also showed improved selectivity profiles compared to PP1 and commonly known inhibitors of SRC and mTOR kinases. Reported, herein, is the discovery of the first sub-nanomolar SRC inhibitor which does not inhibit the kinase ABL and shows excellent properties suitable for further preclinical development.

Background: Radiotherapy is used in the treatment of over 50% of cancer patients and bar surgery, is the most effective cancer intervention. However, in the clinic secondary malignancies have been observed following radiotherapy and in vitro increased cell migration and invasion have been seen following radiation. The Src/FAK signalling pathway is known to play an important role in the metastatic phenotype through its involvement in cell adhesion, migration and invasion and we have previously demonstrated that radiotherapy can activate this pathway along with the phosphoinositide 3-kinase (PI3K) pathway, also associated with tumour metastases and an aggressive phenotype. Using pharmacological inhibitors, we have investigated combination approaches to evaluate whether Src and PI3K targeting is beneficial in a radiotherapy context, especially focusing on metastatic phenotype. We wished to relate pathway activation to cellular phenotype and increase understanding of the metastatic cascade, the processes involved and the signalling pathways taking the lead. Method: Using thyroid carcinoma cell lines FTC133 and 8505c the effects of Src inhibition using AZD0530, FAK inhibition using FAKi and PI3K inhibition using GDC-0941 were studied. The effects of radiotherapy alone, and in combination with the above inhibitors, were also studied. In vitro MTT, apoptosis and clonogenic assays were used to assess cell proliferation and cell survival and scratch assays, cell adhesion and cell spreading assays were used to assess the effects of the drugs on metastatic characteristics. In vivo tumour growth, survival and ex vivo clonogenics were used to measure the effects of AZD0530 and GDC-0941. Western blotting, immunofluorescence and immunohistochemistry was used to observe the effects on pathway activation and protein localisation. Results: Src and FAK inhibition reduced metastatic characteristics of thyroid carcinoma cell lines in vitro such as cell spreading and migration. FAK inhibition showed a greater effect on cell survival by MTT, clonogenic and apoptosis. In the thyroid carcinoma cell lines radiotherapy enhanced the metastatic phenotype. This was seen by enhanced activation of the Src and PI3K pathways, increased migration and invasion in vitro and enhanced tumour metastasis in vivo. By combining Src inhibition with radiation a reduction in metastatic characteristics was observed and by combining PI3K inhibition with radiotherapy radiosensitivity could be improved. With the triple combination of Src and PI3K inhibition with radiotherapy a significant reduction in cell survival was demonstrated in vitro compared to radiation alone and either inhibitor combined with radiation, with a corresponding significant reduction in tumour growth being observed in vivo. With the combination of Src and PI3K inhibition significant reductions in metastatic characteristics were also observed both in vitro and in vivo seen by a reduction in cell migration and tumour metastasis. Finally combined inhibition of the Src and PI3K pathway reduced the radiation enhanced activation of several pathways in vivo including Src and PI3K.Conclusions: Together these results suggest that the Src and PI3K pathways play a role in radiation enhanced metastatic characteristics in thyroid carcinoma and through combined inhibition of the pathway the negative effects of radiation, enhanced migration and invasion, can be inhibited and the cells can be made more radiosensitive. Full characterisation of the pathways involved in radiation induced motility and radioresistance will provide further rationale for combination therapies and provide potential for application of these therapies in the clinic.

La tyrosine kinase cytoplasmique Src est un régulateur essentiel de la croissance et de l’adhésion cellulaires induites par de nombreux stimuli extracellulaires, dont les facteurs de croissance et certains composants de la matrice extracellulaire. La dérégulation de son activité catalytique lui confère des propriétés oncogéniques importantes conduisant à la formation de tumeurs agressives chez l’Homme. La plupart des connaissances actuelles sur sa régulation catalytique repose sur des données structurales de cristallographie qui ont révélé l’importance des interactions intramoléculaires SH2 et SH3-dépendantes dans le contrôle des conformations ouvertes et actives de Src. Les domaines SH3, SH2 et kinase de Src sont associés au domaine SH4 N-terminal d'ancrage à la membrane plasmique via un domaine unique (DU) flexible. Le rôle du DU dans la régulation de Src reste obscur car il est intrinsèquement désordonné et par conséquent, cette région n'a pas été incluse dans les analyses structurales originelles. L'analyse par RMN de l'extrémité N-terminale de Src a révélé une conformation partiellement structurée du DU par le contact de séquences peptidiques spécifiques avec les lipides membranaires et le domaine SH3 (Perez et al, 2013; Maffei et al, 2015). De plus, cette interaction est régulée par phosphorylation des Ser69 et Ser75 localisées à proximité de ce domaine. L’objectif de ma thèse était d’adresser la relevance biologique de ces données structurales en me focalisant sur le rôle d’une partie du DU identifiée comme importante par RMN. J’ai montré que l’inactivation de cette région par des mutations perte de fonction, diminue fortement la signalisation de Src dans les cellules non transformées humaines ainsi que ses propriétés tumorales dans les cellules cancéreuses colorectales métastatiques révélant la pertinence biomédicale du système. Des analyses de protéomique m’ont permis de révéler un mécanisme original par lequel cette région contrôle la capacité de Src à phosphoryler ses substrats. En conclusion, ces données confirment un rôle important du DU sur l'activité et la signalisation de Src et révèlent un rôle critique des domaines désordonnés dans les tumeurs chez l’Homme. L’inhibition de cette région unique par de petites molécules devrait conduire à de nouvelles stratégies thérapeutiques dans le cancer. / The membrane-anchored non-receptor tyrosine kinase Src is involved in numerous signal transduction pathways and hyperactive Src is a potent oncogene and driver of human metastasis. Most of our knowledge on Src kinase regulation relies on structural data, which revealed SH2 and SH3-dependent intramolecular interactions to control active conformations of the protein. The kinase, SH3 and SH2 domains of Src are attached to the membrane-anchored SH4 domain through the flexible unique domain (UD). The role of this UD remains obscure due to its intrinsically disordered properties for which reason it was not included in original structural analyses. Interestingly, membrane-associated intrinsic disordered domains are more prevalent among signaling and cancer-related proteins and they are thought to play critical roles in human disease. NMR analysis of Src N-terminus in the presence of lipids revealed a partially structured conformation of the UD through the contact of a small peptide sequence with membrane lipids and the SH3 domain of the protein (Perez et al, 2013; Maffei et al, 2015). This interaction was regulated by phosphorylation of Ser69 and Ser75 surrounded this central region. The aim of my thesis project was to assess the biological relevance of this structural data. Interestingly, I showed that expression of Src mutants with UD loss of function drastically affected Src activity and signaling in human non-transformed cells as well as Src oncogenic properties in metastatic colorectal cancer cells. This highlights the biomedical relevance of the system. Further proteomic analysis revealed an unsuspected mechanism by which this region controls the Src capacity to phosphorylate specific substrates involved in cancer cell activity. These data support a previously unrecognized important role of the UD on Src activity and signaling and reveals a critical role of intrinsic disordered domains in the regulation of kinase signaling in human disease. Targeting this unique region by small molecules may be of therapeutic value in human cancer.

Ce travail a pour objet d’étude l’émergence d’effets d’affordance dans la schizophrénie. En effet, les troubles de l’action, observés chez les patients schizophrènes, pourraient être la conséquence d'un déficit de l’intégration sensorimotrice, ne permettant pas le couplage entre perception et action. En lien avec les théories de la cognition incarnée et située, nous avons fait l’hypothèse que l’enrichissement de la situation puisse permette l’émergence de ce lien dans cette population. Pour ce faire, nous avons décliné 5 expériences qui utilisent le paradigme SRC (Stimulus-Response-Compatibility). L’expérience 2 se focalise sur l’intégration visuo-spatiale, tandis que les 4 autres portent sur l’effet de potentialisation des actions lors de la perception d’objets manipulables (Tucker & Ellis, 1998), selon différentes conditions: sans amorçage (expérience 1), avec amorçage renforçant le sentiment de propriété des objets (expérience 3) ou les buts de l’action (expérience 4) et avec amorçage moteur (expérience 5). Nos résultats mettent en évidence l’émergence d’un effet de compatibilité visuo-spatiale (expérience 2) et sensorimotrice (expérience 1), dans le groupe de témoins. Toutefois, cet effet n’est pas retrouvé lorsque l’amorçage est incongruent avec les objets présentés (expériences 3 et 4) ou lorsqu’il implique un amorçage moteur (expérience 5). Dans le groupe de patients schizophrènes, bien que l’effet de compatibilité visuo-spatiale soit mis en évidence (expérience 2), l’effet de compatibilité sensorimotrice n’est retrouvé que lorsque la perception des objets est précédée d’une préparation motrice (expérience 5). Ces résultats sont discutés à la lueur des connaissances issues des théories de la cognition incarnée et située, et des travaux centrés sur les troubles de l’action retrouvés dans la schizophrénie. / This thesis studies the emergence of affordance effects in schizophrenia. Indeed, the disorders of action present in schizophrenic patients might be the consequence of deficit in sensorimotor integration, making difficult the link between perception and action. Related to the embodied cognition framework, we hypothesized that the enhancement of context would allow the emergence of this link in this population. Then, we planned 5 experiments using the SRC paradigm (Stimulus-Response-Compatibility). Experiment 2 emphasizes visuo-spatial integration. The four others focus on the effect of potentiation of actions during perception of handling objects (Tucker & Ellis, 1998), depending on different experimental conditions: without priming (experiment 1), with priming reinforcing feeling of being owner of objects (experiment 3) or reinforcing the aims of action (experiment 4) and with motor priming (experiment 5). In the control group, our results show emergence of visuo-spatial (experiment 2) and sensorimotor (experiment 1) compatibility effects. However, this effect is not present when priming is not congruent with presented objects (experiment 3 and 4) or in case of motor priming (experiment 5). In patients, despite of the presence of visuo-spatial compatibility effect (experiment 2), the sensorimotor compatibility effect is present only when perception of objects is preceded by a motor preparation (experiment 5). These results are discussed in reference to the theories of embodiment, and from studies focused on disturbances of action in schizophrenia.

The non-receptor tyrosine kinase Src has been found to be overexpressed and activated in many human cancers, where it has been implicated in changes in cellular proliferation, adhesion, migration, apoptosis, angiogenesis, and tumour growth. In addition, several other members of the Src family have also been implicated in various cancer phenotypes. Our examination of a wide panel of colon, breast, and lung cancer cell lines revealed that not only Src, but also Yes, Fyn, Lyn, and Lck, were expressed at both the mRNA and protein levels in different combinations, and at varying levels, between cell lines. When examined for kinase activity, it was discovered that only a subset of the expressed Src family members had detectable kinase activity within a given cell line. To investigate the involvement of the Src family members in the proliferation, adhesion, migration, and colony forming ability of four selected cancer cell lines, both Src family kinase inhibitors, which inhibit the kinase activity of multiple Src family members, and RNA interference, which selectively decreases the expression of individual proteins, were used. It was found that the involvement of these proteins in all of the cellular processes investigated was cell line-specific, with the greatest effects observed in HT29 cells, which have relatively high Src protein levels and kinase activity. Furthermore, the consequences of Src family member inhibition were also inhibitor specific, as treatment with PP2 and SKI I generally had greater effects on the cellular processes examined than did treatment with SU6656 or SKI II. It was also found that the inhibition of multiple Src family kinases by at least one of the inhibitors generally resulted in greater effects on the cancer cell phenotypes investigated than were observed when the expression of Src, Fyn, or Yes was decreased using RNA interference. This suggests that multiple Src family members may need to be targeted in order to inhibit the increased proliferation, cell-matrix adhesion, migration, and colony forming ability exhibited by cancer cells. The identification of the cancer cell phenotypes in which particular Src family members are involved is of interest, as these proteins are attractive targets for cancer therapy.