Les tumeurs mammaires canines (CMT) sont les plus fréquents néoplasmes chez les chiennes intactes. Aucun consensus n’existe, actuellement, concernant le protocole thérapeutique des tumeurs invasives ou métastatiques. Des études récentes démontrent l’implication du dérèglement de la voie Hippo dans le développement et la progression des CMTs. Les statines, en inhibant la geranylgeranylation (GGylation) protéique, activent la voie Hippo et suspendent les effets transcriptionnels de YAP et TAZ. Cette étude évalue le ciblage thérapeutique de la voie Hippo avec deux statines (atorvastatine et fluvastatine) sur deux lignées cellulaires de CMT (CMT9 et CMT47). Les deux statines ont démontré une cytotoxicité pour CMT9 et CMT47 avec de IC50 de 0,95 µM et 23,5 µM respectivement. Les statines ont également diminué les niveaux des protéines effectrices de la voie, YAP et TAZ, ainsi que les niveaux d’ARNm de trois gènes cibles de YAP-TAZ/TEAD connus pour leur rôle dans la progression du cancer du sein et la chimiorésistance : CYR61, CTGF et RHAMM. De plus, la migration cellulaire de CMT47 et la croissance indépendante de l’ancrage des deux lignées ont été inhibées en présence de statines mais pas l’invasion de la matrice extracellulaire. Enfin, les analyses FACS ont montré une augmentation de l’apoptose et un arrêt du cycle cellulaire. Nos résultats suggèrent que les statines activent la voie Hippo et ciblent différents mécanismes moléculaires et cellulaires dans les CMT in vitro. Les statines agissent sur la voie Hippo et pourraient représenter une approche innovante pour le traitement des tumeurs mammaires canines invasives ou métastatiques. / Canine mammary tumours (CMTs) are the most common neoplasms in intact bitches, and no effective chemotherapeutic options are available for highly invasive and metastatic tumors. Recent studies show the potential involvement of dysregulated Hippo signaling in CMT development and progression. Protein geranylgeranylation (GGylation) is an important post-translational modification for many signaling molecules, and its blockade with statins has been shown to inhibit YAP/TAZ-mediated transcriptional activity via activation of the Hippo pathway. In this study, we sought to determine if protein GGylation represents a valid pharmacological target in CMTs. Two CMT cell lines (CMT9 and CMT47) were evaluated for their sensitivity to atorvastatin and fluvastatin. Results demonstrated statins to be cytotoxic to both cell lines, with IC50 values ranging from 0.95 µM to 23.5 µM. In addition, both statins lowered Hippo pathway effector proteins YAP and TAZ and reduced the mRNA levels of key transcriptional target genes known to be involved in breast cancer progression and chemoresistance (CYR61, CTGF and RHAMM). Moreover, both statins effectively inhibited cell migration in CMT47 and anchorage independent growth in CMT9 and CMT47, but did not modulate matrix invasion. Finally, our FACs results showed that both statins increase apoptosis and promote cell cycle arrests. Taken together, our results indicate that statins activate the Hippo pathway in CMTs and modulate several aspects of CMT’s molecular and cellular behaviours. These findings suggest that targeting the Hippo pathway with statins could be a novel approach for the treatment of invasive, metastatic or inoperable canine mammary gland cancers.

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2013-09-29 23:45:35.395

The paralogous multi-functional adaptor proteins YAP and TAZ are nuclear effectors of the Hippo pathway, a central regulator of developmental organ size control, tissue homeostasis and tumour suppression. YAP/TAZ target the TEAD transcription factor family to promote cell survival and inhibit apoptosis. TEAD proteins contain a DNAbinding domain and a YAP/TAZ interaction domain. PCR analysis of medaka fish TEAD cDNA revealed the presence of alternative TEAD splice-forms with variations at the C-terminus of the DNA-binding domain. Structural analysis indicated the YAPbinding domain of TEAD proteins is folded and globular. NMR spectroscopy showed that the TEAD binding domain of YAP does not contain secondary structure. YAP and TAZ both contain WW domains, which are small protein-protein interaction modules. Two YAP isoforms are known, YAP1 and YAP2 that contain one and two WW domains, respectively. To date, only a single WW isoform of TAZ has been described. PCR analysis of medaka TAZ cDNA identified both single WW and tandem WW isoforms of TAZ. NMR spectroscopy was used to characterise structural, conformational, and peptide binding features of the tandem WW domains from YAP and TAZ. The YAP WW2 solution structure confirms that the domain has the canonical anti-parallel β-sheet WW fold. WW1 of YAP and both WW domains of TAZ undergo conformational exchange. The region linking the two WW domains is flexible and allows interaction of both WW domains with peptides containing single and dual PPxY binding motifs. In addition to YAP and TAZ, tandem WW domains are also present in the core and upstream Hippo pathway proteins Salvador and Kibra. Both proteins contain one atypical WW domain; the tandem WW domains of these two proteins are unstable. Understanding structure and function of Hippo pathway components could contribute to drug development and will also contribute to knowledge of protein folding and interactions.

Trabalho de investigação desenvolvido na Universidade do Porto. Instituto de Ciências Biomédicas Abel Salazar, e no National Institute for Materials Science(Tsukuba, Japan) / Tese de mestrado integrado. Bioengenharia (Biotecnologia Molecular). Faculdade de Engenharia. Universidade do Porto. 2012

This thesis aims to analyse the architectural production in Turkey during the Second World War period with reference to its socio-political contexts, by examining and scrutinizing an architectural periodical published in this period: Yapi. The conceptual framework of this study about the practical and ideological aspects of the discipline are drawn by considering the developments and dynamics of the early Republican Period. In the first chapter of the study after the introduction, the functions of periodical publications for the cultural atmosphere and the architectural periodicals for the discipline of architecture in particular, are examined in the direction of clarifying the reason of choosing a periodical for an architectural analysis of a specific period. In the following part of this chapter, the early Republican architectural periodicals are introduced together with their reasons of establishment and the architectural conditions of the period. Lastly, the focus of this study, Yapi, is introduced with its identity as a periodical by examining its founders, content and publication quality. In the second chapter, firstly architecture of the country throughout the early Republican period is examined by focusing on the significance and effects of the Second World War on Turkey so as to reveal the existing medium that compelled the architects and intellectuals to reconsider their points of view. The second part of this chapter attempts to make a comparative architectural analysis of the period based on the approaches and articles of Yapi by determining the characteristics and objectives of the periodical and demonstrating the main arguments of the editors and authors on contemporary architectural practice and ideology. In the conclusion part, the outcomes of the analysis of Yapi and its arguments on the architecture and contextual developments of the Second World War period are evaluated in correlation with the meaning and place of these struggles in the contemporary medium of architecture.

Thesis (M.A.)--University of Hawaii at Manoa, 1993 / Pacific Islands Studies

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22641号 / 医博第4624号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The role of vascular endothelial growth factor (VEGF) signaling in cancer is well-known in the context of angiogenesis but is also important in the functional regulation of tumor cells themselves. Notably, autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression and drug resistance. Therefore, the first half of this thesis focuses on the mechanism of VEGF-NRP-mediated support of CSCs. Aberrant activity of the Hippo pathway effector YAP and TAZ are associated with breast CSCs and have been shown to confer stem cell-like properties. I found that VEGF-NRP2 signaling contributes to the activation of YAP/TAZ in various breast cancer cells, which mediates a positive feedback loop that promotes mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, which enabled the activity of YAP/TAZ. In complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) β2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, YAP/TAZ-mediated repression of β2-chimaerin sustained Rac1 activity in CSCs. Depletion of β2-chimaerin in non-CSCs increased Rac1 activity, YAP/TAZ activation and mammosphere formation. Analysis of breast cancer patients revealed an inverse correlation between β2-chimaerin and TAZ expression in tumors. These findings highlight an unexpected role for β2-chimaerin in a feedforward loop of YAP/TAZ activation and the acquisition of CSC properties. Given that CSCs have been implicated in therapy resistance and are enriched in triple negative breast cancer (TNBC), which exhibits VEGF-NRP2 signaling, the second half of this thesis focuses on understanding the mechanism by which VEGF-NRP2 contributes to the chemoresistance of TNBC. I discovered that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double strand break repair. Mechanistically, I found that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. I also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to chemotherapy and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ in response to cisplatin. These findings reveal novel roles for VEGF-NRP2 and YAP/TAZ in DNA repair and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ and Rad51 that contributes to resistance to platinum chemotherapy. In summary, this thesis provides novel insight into the roles of autocrine VEGF-NRP2 signaling in breast CSC function and therapy resistance and provides rationale in inhibiting NRP2 for platinum-resistant tumors that are dependent on YAP/TAZ activation.

Vitamine E analogues α-tocopheryl succinate (α-TOS) and mitochondrially targeted vitamine E succinate (MitoVES) are anti-cancer agents from the group of "mitocans", the compounds acting via mitochondria which present a promising invariant target for cancer cell therapy. α-TOS and MitoVES induce apoptosis selectively in various cancer cell types involving generation of reactive oxygen species (ROS). Generated superoxid anion radicals in response to α-TOS and MitoVES are believed to be converted into hydrogen peroxide that is known to activate Mammalian sterile 20-like kinase (Mst1), the central component of Hippo signalling pathway, that presents an universal size control mechanism in all metazoans and its deregulation is linked to tumourigenesis. MitoVES and α-TOS were both reported to activate Mst1 that phosphorylates Forkhead box O1 (FoxO1) transcription factor resulting in its transport to nucleus where induce the expression of pro-apoptotic genes, including NOXA, and thus promote apoptosis. The target of Hippo signalling pathway is transcriptional co- activator Yes-associated protein (Yap) which was found in Drosophila melanogaster to regulate the expression of transcription factor c-Myc which is known as the most prominent human oncogene. This thesis focused on involvement of Hippo signalling...

Sesqiterpene lactones, a group of plant secondary metabolites which include Cnicin from Cnicus benedictus plant, have an anti-proliferative and anti-tumor effect on mammalian cells by activating specific signaling pathways while also generating oxidative stress. These factors combined drive tumor cell apoptosis. A few of these compounds have reached clinical trials and seem to be a promising chemotherapeutics. The focus of this work is to elucidate the effect of cnicin on C-MYC transcription factor and oncoprotein which is overexpressed in majority of tumor tissues, the effect of cnicin on DEAD-box RNAhelicase DDX3 and on the expression levels of several metabolic genes is also studied. Through the use of western blotting, immunodetection and qPCR it was found out, that cnicin is regulating the expression of C-MYC oncoprotein on both transcriptional and translational levels, while also lowering C-MYC protein stability probably through the effect on PIM-2 kinase. Cnicin is not affecting the total amount of DDX3 protein in cells, but it seems it is lowering its degradation rate. The possible transcriptional regulation of DDX3 by cnicin is still not clear and requires further research. With the use of LC-MS quantitative analysis and qPCR, it was found out that cnicin does not affect the metabolism of...