Le développement d'anticorps thérapeutiques a suscité beaucoup d'intérêt au cours des dernières décennies. Plus de 30 d'entre eux ont été approuvés et sont utilisés pour traiter des patients atteints de cancer. Les récepteurs agonistes de TRAIL (DR4 ou DR5) sont surexprimés par les cellules tumorales et capables de déclencher leur mort. Ils représentent donc des cibles idéales. Malgré des résultats encourageants, la plupart des essais cliniques basés sur des anticorps monoclonaux ciblants DR4 ou DR5 ont été interrompus. Cependant, les connaissances actuelles ouvrent des perspectives thérapeutiques de choix pour l'utilisation de tels anticorps en oncologie. Afin de développer de nouveaux anticorps anti-DR4 et anti-DR5 reconnaissant sélectivement les protéines natives d’intérêt, et doués de propriétés antitumorales, nous avons opté pour une approche d'immunisation génétique basée sur des injections hydrodynamiques d'ADN complémentaire. Cette approche nous a permis d'obtenir des réponses humorales significatives, et après fusions des rates correspondantes, de générer 21 anticorps monoclonaux capables de reconnaître spécifiquement et avec une très grande affinité les récepteurs DR4 ou DR5, sous leurs formes natives. Parmi ces anticorps monoclonaux, deux sont doués de propriétés pro-apoptotiques, et quatre sont capables d'accroitre le potentiel pro-apoptotique du ligand TRAIL. Les propriétés antitumorales de l'anticorps anti-DR4 le plus puissant, l’AcM-C#16, ont également été validées in-vivo dans des modèles de xénogreffes.L'ensemble de ce travail démontre, et ce pour la première fois, que la méthode d'immunisation ADN par injection hydrodynamique peut être utilisée pour générer des anticorps monoclonaux thérapeutiques efficaces ciblant des récepteurs de la superfamille du TNF. Au-delà du système TRAIL, cette approche d'immunisation, peu exploitée, pourrait ouvrir de nouvelles perspectives thérapeutiques en l'adaptant à de nouvelles cibles. / Development of therapeutic antibodies has attracted many interests in recent decades. More than 30 of them have been approved and are used to treat cancer patients. TRAIL agonist receptors (DR4 or DR5) are overexpressed by the tumour cells and are able to trigger their death. Therefore, they represent ideal targets. Despite encouraging results, most clinical trials based on monoclonal antibodies targeting DR4 or DR5 have been discontinued. However, current knowledge opens therapeutic perspectives of choice for the use of such antibodies in oncology. In order to develop new anti-DR4 and anti-DR5 antibodies recognizing selectively the native form of the proteins of interest, and endowed with antitumor properties, we have chosen to perform a genetic immunization approach based on hydrodynamic injections of complementary DNA. This approach allowed us to obtain significant humoral responses, and after fusions of the corresponding spleens, to generate 21 monoclonal antibodies capable of recognizing specifically and with very high affinity DR4 or DR5 receptors, in their native forms. Of these monoclonal antibodies, two are display pro-apoptotic properties, and four are capable of enhancing TRAIL pro-apoptotic potential. The antitumor properties of the most potent anti-DR4 antibody, mAb-C16, have also been validated using in-vivo xenografts models.Altogether this work demonstrates, for the first time, that the DNA immunization hydrodynamic injection method can be used to generate therapeutically effective monoclonal antibodies targeting TNF superfamily receptors. Beyond the TRAIL system, this immunization approach, scarcely exploited, could open new therapeutic perspectives by adapting it to new targets.

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[EN] Abstract Fruit set is the transition from a quiescent ovary of the flower to a developing fruit after pollination. This process is associated with the content of gibberellin (GAs) and auxins in the ovary. Therefore the development of parthenocarpic fruit (seedless fruit) can be induced by applying both kinds of hormones. In tomato it has been described that auxin action during fruit set is mediated by GAs, by inducing different biosynthesis pathway genes, among them SlGA20ox, which codify important enzymes for GAs biosynthesis regulation. The SlGA20ox gene family is composed of 4 genes, and SlGA20ox1 seems to play a key role in fruit set. In order to deepen the understanding of this process, it has been investigated in the ovary of tomato: a) the site of expression of SlGA20ox1 and its regulation; b) the localization of IAA (auxin). For this, we have obtained transgenic lines of the tomato Micro-Tom (MT) cultivar and various hormonal mutants (procera, 35S::CsGA20ox1 Dwarf, entire and dgt) with the transgene pSlGA20ox1::GUS. Transgenic lines do not differ phenotypically from the original lines and they have been used to analyze the expression of SlGA20ox1, using the GUS gene as reporter. Localization of SlGA20ox1 transcripts was also investigated in situ. SlGA20ox1 expression in ovary after pollination was located mainly in the funiculus, placenta and embryo. Auxin and brassinosteroids (BR) increased the expression of SlGA20ox1. On the other hand, auxin location in the ovary was investigated using the transgenic line DR5::GUS (expressing the auxin-response gene DR5), and analyzing directly the IAA content by immunolocalization. In pollinated ovaries, GAs and auxin co-localize in the egg (embryo and embryo-sac) and placenta, supporting the hypothesis that both hormones interact during fruit set. Phenotypic analysis of the mutants used in this work showed that GAs inhibit the development of axillary buds, through the DELLA protein. This inhibitory effect is reversed, at least partially, by BR. Furthermore, the introduction of wild gen Dwarf in MT (normalizing its content of BR) increases plant height, but does not extend its vegetative cycle, nor increases parthenocarpy in MT. / [ES] Resumen La fructificación, paso del ovario en reposo a fruto en crecimiento tras la polinización, asociada al aumento de contenido de giberelinas (GAs) y auxinas en el ovario, es un proceso clave para la producción. Por ello se puede inducir también el desarrollo de frutos partenocárpicos (sin semillas) aplicando ambos tipos de hormonas. En tomate se ha descrito que parte de la acción de las auxinas en la fructificación está mediada por GAs, induciendo distintos genes de la ruta biosintética, entre ellos los que codifican SlGA20ox, enzimas importantes para la regulación de la síntesis de GAs. De los 4 genes que forman la familia, SlGA20ox1 parece ser clave en la fructificación. Con objeto de profundizar en el conocimiento de este proceso, se ha investigado en el ovario de tomate: a) la localización de la expresión de SlGA20ox1 y su regulación; b) la localización de IAA (auxina). Para ello, se han obtenido líneas trangénicas del cultivar Micro-Tom (MT) de tomate y diversos mutantes hormonales (procera, 35S::CsGA20ox1 Dwarf, dgt y entire) con el transgén pSlGA20ox1::GUS. Las líneas transgénicas no difieren fenotípicamente de las líneas originales y se utilizaron para analizar la expresión de SlGA20ox1 con el gen delator GUS. Se investigó además la localización de los transcritos de SlGA20ox1 in situ. La expresión de SlGA20ox1 en el ovario tras la polinización se localizó, principalmente, en el funículo, la placenta y el embrión. Tanto las auxinas como los brasinosteroides (BR) aumentaron la expresión de SlGA20ox1. Por otro lado se investigó la localización de auxinas en el ovario usando una línea transgénica DR5::GUS (que expresa el gen de respuesta a auxinas DR5), y analizando también directamente el contenido de IAA mediante inmunolocalización. En ovarios polinizados, la localización de las GAs y auxinas coincidió en el óvulo (embrión y saco embrionario) y la placenta, apoyando la hipótesis de que ambas hormonas interaccionan durante la fructificación. El análisis fenotípico de los mutantes utilizados mostró que las GAs inhiben el desarrollo de los brotes axilares por GAs, a través de la proteína DELLA. El efecto inhibidor es revertido, al menos parcialmente, por BR. Por otro lado, la introducción del gen silvestre Dwarf en MT (normalizando así su contenido en BR) aumenta la altura de la planta, pero no prolonga su ciclo vegetativo, ni aumenta la capacidad partenocárpica de MT. / [CAT] Resum La fructificació, pas de l'ovari en repòs a fruit en creixement després de la pol·linització, associada a l'augment de contingut de giberelines (GAs) i auxines en l'ovari, és un procés clau per a la producció. Per això es pot induir també el desenvolupament de fruits partenocàrpics (sense llavors) aplicant tots dos tipus d'hormones. En el tomàquet s'ha descrit que part de l'acció de les auxines en la fructificació està intervinguda per GAs, induint diferents gens de la ruta biosintètica, entre ells els que codifiquen SlGA20ox, enzims importants per a la regulació de la síntesi de GAs. Dels 4 gens que formen la família, SlGA20ox1 sembla ser clau en la fructificació. A fi d'aprofundir en el coneixement d'aquest procés, s'ha investigat en l'ovari del tomàquet: a) la localització de l'expressió de SlGA20ox1 i la seva regulació; b) la localització de l'IAA (auxina). Per a això, s'han obtingut línies transgèniques de conrear Micro-Tom (MT) de tomàquet i diversos mutants hormonals (procera, 35S::CsGA20ox1 Dwarf, dgt i entire) amb el transgèn pSlGA20ox1::GUS. Les línies transgèniques no difereixen fenotípicamente de les línies originals i es van utilitzar per analitzar l'expressió de SlGA20ox1 amb el gen delator GUS. Es va investigar, a més, la localització dels transcrits de SlGA20ox1 in situ. L'expressió de SlGA20ox1 en l'ovari després de la pol·linització es va localitzar, principalment, en el funicle, la placenta i l'embrió. Tant les auxines com els brasinosteroides (BR) van augmentar l'expressió de SlGA20ox1. D'altra banda es va investigar la localització d'auxines en l'ovari usant una línia transgènica DR5::GUS (que expressa el gen de resposta a auxines DR5) i analitzant també directament el contingut de IAA mitjançant immunolocalització. En ovaris polinitzats, la localització de les GAs i auxines va coincidir en l'òvul (embrió i sac embrionari) i la placenta, refermant la hipòtesi que ambdues hormones interaccionen durant la fructificació. L'anàlisi fenotípic dels mutants utilitzats va mostrar que les GAs inhibeixen el desenvolupament dels brots axil·lars per GAs, a través de la proteïna DELLA. L'efecte inhibidor és revertit, almenys parcialment, per BR. D'altra banda, la introducció del gen silvestre Dwarf en MT (normalitzant així el seu contingut de BR) augmenta l'alçada de la planta, però no perllonga el seu cicle vegetatiu, ni augmenta la capacitat partenocàrpica d'MT. / Gallego García, M. (2016). Papel de la GA 20-oxidasa en la fructificación del tomate [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61958 / TESIS

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Renal Cell Carcinoma (RCC) has low 5 year survival rate and is resistant to radiation and chemotherapy. Phenethyl Isothiocyanate (PEITC) is a naturally occurring phytochemical that has a variety of anti-cancer properties. Here we explore two anti-cancer properties of PEITC: induction of apoptosis and induction of cell cycle arrest in RCC cells and the underlying mechanisms. We used two human RCC cell lines Caki-1 and Caki-2. Survival and cell proliferation was assayed using Calcein AM. Annexin V staining was used to measure apoptosis. Caspase-3/7 induction was measured using a fluorescent substrate. Cell cycle was studied using Propidium Iodide staining. DNA damage was determined using phospho [gamma]-H2AX antibody. Protein expression and phosphorylation was determined using immunoblotting. PEITC significantly reduced survival of Caki-1 and Caki-2 cells and inhibited their proliferation as determined by Calcein AM. 15 and 20 [mu]M PEITC induced apoptosis in both cell lines and induced caspase-3/7 activity. Western blot analysis revealed caspase-8, caspase-9 and Bid cleavage as well as upregulation of the death receptors Fas and DR5. Lower doses (up to 10 [mu]M) arrested Caki-1 cells in G2/M phase, and this was associated with increased p38 and MK2 (Thr334) phosphorylation. The p38 inhibitor SB203850 inhibited this G2 arrest induced by PEITC. 15 and 20 [mu]M PEITC treatment resulted in increased [gamma]-H2AX phosphorylation suggesting DNA damage, but this was completely blocked by caspase inhibitor. In summary, our study shows that PEITC induces apoptosis in Caki-1 and Caki-2 cells by upregulating Fas and DR5 and activating the downstream apoptosis cascade. PEITC does not cause direct DNA damage to the cells; the observed DNA damage is a result of the apoptotic process and is blocked by caspase inhibitor. PEITC induces G2/M arrest in Caki-1 cells and the mechanism involves p38 phosphorylation which activates MK2. Inducing cell cycle arrest and apoptosis may play an important role in the anti-cancer properties of PEITC. Fully understanding the mechanism by which PEITC induces apoptosis and cell cycle arrest in RCC cells may lead to development of novel chemotherapeutic drugs against RCC. / text

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text