Die Mehrheit chronischer Nierenerkrankungen wird durch glomeruläre Defekte hervorgerufen. In dieser Arbeit wurde deshalb im Mausmodell die Bedeutung der Kollagenrezeptoren DDR1 (Discoidin Domain Rezeptor 1) und ITGA2 (Integrin Alpha 2) in der Pathogenese von glomerulären Nierenerkrankungen untersucht. Von zentralem Interesse waren neben der Betrachtung des renalen Phänotyps, die Analyse der glomerulären Basalmembran sowie die Prüfung auf Vorhandensein nierenschädigender Faktoren. Zur Orientierung angefertigte H.E.-Färbungen waren lichtmikroskopisch unauffällig, jedoch ließ sich mittels Gelelektrophorese eine Mikro-, Makro- und Albuminurie mit einem Maximum zum Zeitpunkt von 100 Lebenstagen nachweisen, die mit 200 Tagen wieder stark sank. Auf dem Boden der nierenschädigenden Proteinurie, zeigten die Western-Blot-Analysen das Vorhandensein der Zytokine TGF-ß und CTGF auf. Die zur Detektion von Narbengewebe durchgeführte Fibronektinfärbung, erbrachte keinerlei weiterführende Anhaltspunkte. In der Elektronenmikroskopie ließ sich vereinzelt eine Mehrschichtung der GBM nachweisen, was als Ausreifungsstörung interpretiert wurde. Der Wegfall der beiden Kollagenrezeptoren ITGA2 und DDR1 scheint somit die Interaktion der Podozyten mit der GBM zu stören. Dies hat eine Proteinurie zur Folge. In Folge dessen werden profibrotische Zytokine sezerniert. Das Fehlen der beiden Kollagenrezeptoren DDR1 und ITGA2 führte jedoch nicht zur Ausbildung einer renalen Fibrose, wie in der Fibronektin-Färbung gezeigt werden konnte. Gross und Girgert zeigten, dass nierenkranke Mäuse nach dem Verlust von DDR1 oder ITGA2 einen verzögerten Verlauf der Nierenfibrose entwickelten. Vielversprechend scheinen Untersuchungen z.B. am Mausmodell Col4A3/DDR1/ITGA2 -/- oder an einer diabetischen ITGA2/DDR1 -/- Maus. Gesetzt dem Fall, dass eine renale Fibrose im Vergleich zum Einzelknockout noch später eintritt, eignen sich diese beiden Kollagenrezeptoren als therapeutisches Ziel. Aktuell stehen nur wenige nephroprotektive Medikamente, wie ACE-Hemmer, zur Verfügung. Anti-Integrine und Inhibitoren gegen Tyrosinkinase-Rezeptoren, wie DDR1, haben bereits Einzug in den klinischen Alltag gehalten und stellen eventuell einen wirksamen Ansatzpunkt zur Verhinderung einer renalen Fibrose dar.

Antagonism of the activin receptor signaling pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous research has shown that antagonism of activin signaling promotes muscle growth, attenuates muscle wasting, and restores function in both wild type and diseased animals. Our laboratory has recently developed a novel small molecule (SGI-1252) that inhibits activin downstream (i.e., Smad2/3 phosphorylation) signaling. Purpose: In this study we determined how eight weeks of orally administered SGI-1252 affected TGF-ß signaling, whole body mass, individual limb muscle mass, and muscle fiber cross sectional area (CSA). Methods: Wild-type (WT) mice were treated with SGI-1252 or a vehicle control (VC) via oral gavage (400 mg/kg 3 times per week) for 8 weeks. Body mass was measured twice per week during the 8-week treatment period. At the end of the treatment period, gastrocnemius and tibialis anterior (TA) muscles were excised, weighed, and prepared for histological and biochemical analyses. Results: Following 8 weeks of treatment, there was no difference in weight gain between SGI-1252 (24.8 ± 1.8g) and VC treated mice (23.2 ± 1.5g) (p = 0.06). Gastrocnemius whole muscle mass was significantly greater in the SGI-1252 treated group relative to the VC treated mice (139.6 ± 12.8 mg vs 128.8 ± 14.9 mg) (p = 0.04), although when normalized with body mass there was no difference in gastrocnemius mass. For the TA muscle, there were no significant differences in whole muscle mass between SGI-1252 and VC groups, yet TA muscles in the SGI-1252 treated group had a reduced muscle fiber CSA compared to controls (621 ± 44 µm2 vs 749 ± 36 µm2) (p = 0.0005). There was a statistical trend of decreasing Smad2 phosphorylation in the SGI-1252 treated TA muscles (mean SGI-1252 = 0.668 vs VC = 0.848) (p = 0.06), and no significant differences in Smad2 phosphorylation in the gastrocnemius. Conclusions: Contrary to our hypothesis, 8 weeks of orally administered SGI-1252 was not effective in promoting increases in whole body mass, limb whole muscle mass, or myofiber cross sectional area. This may be due to the inability of SGI-1252, at the administered dose, to effectively decrease signaling downstream of the activin receptor. Clearly, studies using a wider range of doses and delivery methods will be needed to ascertain the efficacy of SGI-1252 as a potential therapeutic.

Au début des années 2000, il a été montré que le développement des métastases était fortement limité par un phénomène de dormance cellulaire. En effet, après s’être extravasées dans un organe distant, la plupart des cellules tumorales disséminées qui survivent entrent immédiatement dans un état quiescent réversible mais durable sur le long terme, qui est qualifié de dormant. Si le phénomène de dormance permet de limiter le taux de développement de métastases, il est aussi à l’origine de récidives métastasiques tardives, car les cellules dormantes peuvent persister longtemps et sont résistantes à la chimiothérapie. Il est donc important d’analyser les mécanismes moléculaires régulant la dormance des cellules tumorales. Mais cette analyse est difficile à réaliser in vivo du fait de la relative grande rareté des cellules cancéreuses disséminées. Récemment, mon équipe d’accueil a observé que la clonogénicité in vitro des cellules de cancer de la prostate peut être fortement régulée par un phénomène de dormance. Celle-ci est induite lorsque les cellules sont cultivées à la faible densité cellulaire et dans un milieu légèrement hypertonique comme le DMEM. L’objectif de mon travail de thèse était d’analyser les mécanismes moléculaires régulant la dormance des cellules de cancer de la prostate in vitro.Dans un premier temps, nous avons mis en évidence que la dormance nécessite l’activation conjointe de la voie de signalisation BMP/TGF-ß et d’un stress oxydatif. La faible densité cellulaire joue un rôle capital dans l’établissement de la dormance en pré-activant la voie du BMP/TGF-ß et en sensibilisant la cellule au stress oxydatif. L’amplification de ces effets par l’hypertonicité explique l’induction du phénomène de dormance. Nous avons étendu la portée de ces observations en montrant que les androgènes à des concentrations proches des niveaux physiologiques permettent d’induire un phénomène de dormance très similaire, reposant lui-aussi sur l’activation des voies de signalisation du BMP/TGF-ß et du stress oxydatif. De manière intéressante, nous avons confirmé qu’une fois que la dormance est établie, elle se maintient d’une manière autonome. Ainsi, un traitement transitoire par des androgènes s’avère suffisant pour induire la dormance qui se maintient après la remise en culture dans un milieu dépourvu d’androgènes ajoutés. Par ailleurs, nous avons identifié CDKN1A (p21CIP1/WAF1) comme un régulateur de la dormance principalement régulé par le stress oxydatif. Sa surexpression à faible densité cellulaire suffit à induire un phénomène de dormance caractéristique.En conclusion, nous avons montré que la dormance des cellules de cancer de la prostate est régulée par les voies de signalisation du BMP/TGF-ß et du stress oxydatif qui semblent être interconnectées au travers d’une boucle de régulation auto-entretenue. Les androgènes pourraient constituer des inducteurs de dormance efficaces in vivo pour limiter le développement des métastases, ce que nous voudrions tester dans un modèle de dissémination métastasique simplifié chez la souris obtenu par injection intracardiaque de cellules cancéreuses. Par ailleurs, notre modèle prédit que les CTCs (Cellules Tumorales Circulantes dispersées dans la circulation sanguine) pourraient être induites à entrer en dormance avant même de s’extravaser du milieu sanguin du fait des conditions pro-oxydantes régnant dans ce milieu, ce qui pourrait être testé en étudiant si des inhibiteurs du stress oxydatif et/ou des voies du BMP/TGF-ß favorisent la prolifération de CTCs purifiées et mises en cultures. / In the early 2000s, development of metastases was shown to be strongly limited by a phenomenon of cellular dormancy. Indeed, after extravasation in a distant organ, most of the disseminated cancer cells that survive enter into a reversible quiescent that is called dormant state. The phenomenon of dormancy is also the cause of late metastatic recurrences because dormancy confers long-term cellular survival and resistance to chemotherapy. Thus, it is important to analyze the molecular mechanisms regulating cancer cell dormancy. However, in vivo studies are difficult due to the extreme scarcity of disseminated cancer cells. Recently, T. Tchenio et al observed that clonogenicity of prostate cancer cells was strongly regulated by a dormancy phenomenon in vitro. A dormant state was induced when cells are cultured at low density in a slightly hypertonic medium, such as DMEM. The aim of my thesis was to analyze the molecular mechanisms regulating the dormancy of prostate cancer cells in vitro.We first demonstrated that dormancy required a combined activation of both BMP/TGF-ß and oxidative stress signaling pathways. Low cell density plays a key role in dormancy establishment by priming both signaling pathways. Hypertonicity induced dormancy through further amplifying these signaling pathways. We extended the biological relevance of our observation by showing that a closely related phenomenon could be induced by androgens at concentrations close to physiologic levels. Androgen-induced dormancy also relied on activation of the BMP/TGF-ß and oxidative stress signaling pathways. Interestingly, we observed that once dormancy was established, it was maintained autonomously since a transient treatment with androgen was sufficient to induce a dormant state that was self-sustained after withdrawal of exogenous androgens. Besides, CDKN1A (p21 CIP1/WAF1) was identified as a dormancy regulator modulated by oxidative stress. We showed that its transient overexpression at low cell density was sufficient to induce a dormancy phenomenon that mimicked those induced by hypertonicity or androgens.In conclusion, we showed that dormancy of prostate cancer cells was regulated by BMP/TGF-ß and oxidative stress signaling pathways that seemed to be interconnected through a self-sustained regulation loop. Androgen may constitute an effective inducer of dormancy in vivo to limit metastases development. Therefore, we would like to test its effects in vivo in a simplified model of metastatic dissemination in mouse relying on intracardiac injection of cancer cells. Furthermore, our model allowed us to predict that CTCs (Circulating Tumor Cells dispersed in bloodstream) could enter into a dormant state before their extravasation from blood vessels due to the pro-oxidant conditions in this environment. This prediction could be tested by studying whether oxidative stress and BMP/TGF inhibitors could promote proliferation of purified CTCs in vitro.

Cells adhere to each other and to the extracellular matrix (ECM) through protein molecules on the surface of the cells. The breaking and forming of adhesive bonds, a process critical in cancer invasion and metastasis, can be influenced by the mutation of cancer cells. Several molecules have been reported to play a crucial role in cellular adhesion and proliferation, and eventually in cancer progression, with TGF-β being one of the most important. In this thesis, we propose a general framework to model cancer cells movement and aggregation, in response to nonlocal social interactions (that is, attraction towards neighbours that are far away, repulsion from those that are near by, and alignment with neighbours at intermediate distances), as well as other molecules' effect, e.g., TGF-β. We develop nonlocal mathematical models describing cancer invasion and metastasis as a result of integrin-controlled cell-cell adhesion and cell-matrix adhesion, for two cancer cell populations with different levels of mutation. The models consist of nonlinear partial differential equations, describing the dynamics of cancer cells and TGF-β dynamics, coupled with nonlinear ordinary differential equations describing the ECM and integrins dynamics. We study our models analytically and numerically, and we demostrate a wide range of spatiotemporal patterns. We investigate the effect of mutation and TGF-β concentration on the speed on cancer spread, as well as the effect of nonlocal interactions on cancer cells' speed and turning behaviour.

Bone repair is modulated by different stimuli. There is evidence that the Transforming Growth Factor-beta (TGF-β) super-family of cytokines have significant effects on bone structure by regulating the replication and differentiation of chondrocytes, osteoblasts and osteoclasts. There is also significant evidence that interactions with extracellular matrix molecules also influence cell behaviour. This study aimed at determining the role of the TGF-βs, Collagen type I, Fibronectin and Laminin in bone cell behaviour. To do this MG63 bone cells were used to examine cell adhesion and alignment to different micro-contact printed ECM protein patterns of different widths. The study also aimed at examining how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell surface interactions, cell morphology, cell proliferation and integrin expression. Finally, this study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence ECM secretion and integrin expression. 5, 10, 25, 50 and 100μm wide repeat gratings of Collagen type I, Fibronectin and Laminin patterns were stamp patterned onto glass slides and plated with MG63 cells at 50,000 cells per coverslip. Cells on the fibronectin pattern attached and elongated soon after seeding, but did not adhere readily to collagen and laminin and appeared more rounded until 18hrs after seeding. Cells aligned significantly well on the 50μm and 100μm wide fibronectin patterned coverslips with mean angles of alignment ~7.87° ± 3.06SD and 6.45° ± 5.08SD, respectively, compared to those with smaller width (p<0.001). In comparison, cells aligned less readily to the other two ECM proteins, showing optimal alignments of 9.66° ± 4.18SD and 14.36° ± 1.57SD to the 50μm wide collagen and laminin patterns, respectively. Differences in cell length mirrored those of alignment, with cells acquiring the greatest length when showing the greatest degree of alignment. The results indicate that MG63 cells responded significantly better to 50 and 100μm wide fibronectin patterns compared to those with smaller width (p<0.001) indicating that the cells may attach mostly via fibronectin specific integrins. Cell surface attachment was examined via a trypsinisation assay in which the time taken to trypsinise cells from the surface provided a means of assessing the strength of attachment. The results indicated that treatment with the solvent (HCl), TGF-β1, 2 and 3 all decreased cell attachment, but this effect was significantly greater in the case of HCl and TGF-β3 (p<0.001). However, there were significant differences in trypsinisation rates between HCl and TGF-β3 (p<0.001). The wound healing response to the TGF-βs and their solvent/carrier was also investigated in 300μm ± 10-30μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The results indicated that TGF-β3 and HCl significantly enhance wound closure when compared against negative controls, TGF-β1 and TGF-β2 treatment (p<0.001). It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p<0.001). Experiments were performed to determine if the HCl effects on wound closure were dose dependent. Cells were incubated with 20μM, 40μM, 80μM and 160μM concentrations of HCl prior to wounding and wound closure rates were recorded. Wound closure was dependent on HCl dose with the 80μM and 160μM concentrations inducing increases in wound closure rates that were both significantly greater than those induced by 20μM, 40μM and control treatments (p<0.001). However, there were significant differences in wound closure between the 80μM and 160μM treatment groups after 30hrs of treatment (p<0.001). The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. The results suggest that cell morphology changes were observed significantly more in cells treated with TGF-β(2+3) and TGF-β(1+3) (p<0.001). Any cell treated with TGF-β1, TGF-β(1+2) and TGF-β(1+2+3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2+3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control due to perhaps its inhibitory effect in contact with human bone cells. Immunostaining indicated that treatment with TGF-β3 significantly promoted the secretion of collagen type I and anti-human fibronectin in addition to integrin (α3 and β1) expression. Statistically TGF-β3 and their combinations showed significant differences in number of cells stained for collagen type I, anti-human fibronectin, α3 and β1 integrin. Any cell treated with TGF-β1 or any combination with TGF-β1 showed significantly lower cell number stained with the same proteins and integrins (p<0.001). Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. WSPR images revealed guided cells with high contrast band like structures at the border of cells distal to the edge of guidance cue to which they aligned and with less concentrically formed band like features across the cell body. It is believed that the high contrast features are associated with the formation of focal contacts on the edge of the cells distal to the edge of fibronectin patterns, which suggests that cell guidance is aided by a decrease in cell attachment along a guidance feature. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In the case of TGF-β1 treated cells the bright high contrast regions were intense but only arranged around the periphery of the cell. In TGF-β2 and TGF-β3 cells the bright contrast regions were weaker but again mostly localised around the periphery. These findings supported the earlier trypsinisation results.

Les vaisseaux sanguins sont composés de cellules enchâssées dans la matrice extracellulaire (MEC). Certaines interactions entre les cellules et les composés de la MEC sont impliquées dans plusieurs changements pathologiques et physiologiques au niveau de la paroi artérielle. L'hypertension systolo-diastolique provoque deux formes de remodelage vasculaire : hypertrophique dans les artères de conductance et eutrophique dans les artères de résistance. Les mécanismes régissant le remodelage eutrophique ne sont pas encore bien éclaircis. Afin de les étudier, nous avons utilisé un modèle d'hypertension systolodiastolique induite par l'inhibition de la synthèse de monoxyde d'azote. Nous avons démontré que la contribution des protéines de désadhésion semblait différer d'un type de remodelage à l'autre et que les métalloprotéinases matricielles (MMPs) ne jouaient pas un rôle crucial dans le développement du remodelage eutrophique, contrairement au remodelage hypertrophique. Au cours du vieillissement, on observe des modifications de la paroi vasculaire des artères de conductance, augmentant leur rigidité : une fragmentation des fibres élastiques, leur calcification (élastocalcinose), une augmentation des liens covalents entre les protéines de la MEC et une fibrose. Cette rigidité conduit au développement de l'hypertension systolique isolée. Afin d'évaluer l'implication des MMPs dans la fragmentation de l'élastine et leur rôle dans le développement de l'élastocalcinose, nous avons utilisé un modèle animal d'élastocalcinose basé sur l'inhibition de la maturation de la matrix Gla protein (MGP), une protéine anti-calcifiante, par la warfarine. Nous avons observé une augmentation rapide et transitoire de l'activité de la MMP-9, suivie de l'activation du transforming growth factor-ß (TGF-ß). L'inhibition de l'activité des métalloprotéinases et de TGF-ß a permis de prévenir l'élastocalcinose. Par ailleurs, l'élastocalcinose est accélérée par le diabète. Cette accélération est reliée à la durée et à la sévérité du diabète. Or, ce dernier est associé à une augmentation de la synthèse des produits avancés de glycation (AGEs) pouvant former des liens covalents iv entre les protéines de la MEC. Ne disposant pas de modèle animal pour étudier l'implication des AGEs dans l'élastocalcinose associée au diabète, nous en avons créé un. Dans ce modèle, le diabète est induit par une diète riche en lipides et une injection de streptozotocine (30 mg/kg/jr). L'élastocalcinose est provoquée par l'inhibition de la maturation de la MGP. Nous avons observé une accélération de l'élastocalcinose, liée à la durée du diabète, et une accumulation de AGEs dans la paroi des artères fémorales. L'utilisation de pyridoxamine, un inhibiteur de la formation des AGEs, et de l'ALT711, un briseur des liaisons covalentes formées par les AGEs, a permis de prévenir et de limiter, respectivement, la calcification dans ce modèle. En outre, nous avons montré que la stimulation du récepteur des AGEs (RAGE), pouvait être impliquée dans l'élastocalcinose et élucidé certains éléments de signalisation dans ce processus. Ces travaux ont fait émerger le rôle des MMPs, de TGF-ß et des produits avancés de glycation dans le remodelage hypertrophique et l'élastocalcinose associée ou non au diabète. Ceux-ci sont reliés à l'apparition de l'hypertension systolo-diastolique et systolique isolée, respectivement. L'inhibition de ces acteurs pourrait constituer de nouvelles thérapies anti-hypertensives.

[Truncated abstract] Fibrosis is a feature of many pulmonary conditions, including idiopathic pulmonary fibrosis (IPF), which is characterised by the accumulation of fibroblasts/myofibroblasts and excessive deposition of collagen. IPF is a disease of unknown aetiology that is unresponsive to current therapy and is typically fatal. The inflammatory cytokine interleukin (IL)-6 is elevated in patients with IPF and recent studies have shown that IL-6-induced signalling is altered in lung fibroblasts from patients with IPF. IL-6 belongs to the gp130 cytokine family, which is a group of ten structurally related cytokines, that all require the membrane bound glycoprotein gp130 to activate intracellular signalling pathways. Gp130 activates intracellular signalling through the Shp2-ERK1/2 and STAT1/3 pathways to mediate cellular activities. This thesis tests the hypothesis that gp130-mediated signalling is dysregulated in the development and progression of pulmonary fibrosis. To address this hypothesis, I assessed the role of gp130-mediated signalling in a mouse model of bleomycin-induced lung fibrosis. This thesis utilised two novel gp130 mutant mice strains with directed and enhanced gp130-mediated Shp2-ERK1/2 (gp130¿STAT/¿STAT) or STAT1/3 (gp130757F/757F) signalling. I observed complete protection from fibrosis in gp130¿STAT/¿STAT mice up to 60 days after bleomycin treatment and profound fibrosis in gp130757F/757F mice compared to wt controls. The enhanced fibrosis observed in gp130757F/757F mice was diminished by monoallelic deletion of STAT3 (gp130757F/757F;STAT3+/-), identifying gp130-STAT3 signalling as a novel promoter of lung fibrosis. ... In addition, IL-6/11 activation of gp130-mediated signalling modulated transforming growth factor (TGF)-ß-induced effects on adult fibroblast proliferation and myofibroblast differentiation. Interaction between IL-6/11 and TGF-ß1 on fibroblast proliferation was dependent on both the gp130-ERK1/2 and gp130-STAT1/3 pathways. Loss of either pathway abrogated the effects of IL-6 and IL-11 on TGF-ß1- 4 induced fibroblast proliferation. However, it was clear that gp130-STAT3 signalling inhibited TGF-ß1-induced myofibroblast differentiation of primary lung fibroblasts. The inhibition of myofibroblast differentiation was associated with gp130-STAT3 dependent inhibition of TGF-ß1-induced Smad3 phosphorylation. These results indicate that IL-6 and IL-11 promote myofibroblastic differentiation of lung fibroblasts, while gp130-STAT3 signalling inhibits TGF-ß1-induced Smad3 phosphorylation and myofibroblastic differentiation of lung fibroblasts While the pathogenesis of IPF is unknown, it is believed that excessive collagen deposition, aberrant fibroblast behaviour and an inflammatory response are critical to the progression of this disease. It has been shown here that IL-6 family cytokines mediate the development and progression of bleomycin-induced lung fibrosis by increasing collagen synthesis, fibroblast proliferation, myofibroblast differentiation and inflammation through gp130-STAT3 signalling. This thesis has demonstrated that differential activation of cytoplasmic signalling pathways by a membrane bound receptor can have a profound effect on pulmonary responses to injury. Furthermore, this thesis is the first study to identify the gp130-STAT3 pathway as a therapeutic target in the treatment of IPF.

Die Notwendigkeit von Bx42 für Drosophila Entwicklung und seine Beteiligung an unterschiedlichen zellulären Prozessen wurde mit Hilfe von RNA Interference (RNAi) demonstriert. Das ubiquitäre Ausschalten oder die Reduktion der Bx42 Expression mittels RNAi führte dabei zu embryonaler Letalität. Weiterhin führte eine gewebespezifische Induktion von Bx42 in Abhängigkeit der verwendeten Treiberlinien bei unterschiedlichen Temperaturen zu mehreren verschiedenen adulten Phänotypen. Diese Phänotypen waren die Grundlage für die Annahme, dass Bx42 eine Rolle in der Regulation mehrerer verschiedener Zellsignalwege spielt. In der Tat interagiert Bx42 mit den Proteinen des Notch-Signalweges Suppressor of hairless [Su(H)] und Notch intracellular domain (N-IC). Zusätzlich werden bei einer Verminderung von Bx42 die Notch Zielgene cut (ct) und enhancer of split m8 [e(Spl)m8] reprimiert (Negeri et al., 2002). In dieser Arbeit wurde die Beteiligung von Bx42 am TGF-ß/Dpp Signalweg untersucht. Es wurde gezeigt, dass Bx42 mit den TGF-ß/Dpp-Signalweg Proteinen Mad und Medea sowohl in vitro als auch in vivo interagiert. Die dabei verwendeten Methoden waren das Hefe-Zwei Hybrid-Sytem und Ni-NTA-Pulldown-Assays. Domänen der Smad Proteine (Mad und Medea), die für die Interaktion mit Bx42 notwendig sind, wurden mit Hilfe von Deletionskonstrukten untersucht. Es konnte gezeigt werden, dass die stark konservierte MH2-Domäne dieser Proteine für die Interaktion notwendig ist. Zudem belegten Versuche die genetische Interaktion zwischen Bx42 und Medea, in denen ein Bx42-RNAi-Phänotyp durch die gleichzeitige Überexpression von Medea gerettet werden konnte. Es ist bekannt, dass das humane Bx42-Homolog Skip sowohl mit den Proteinen Smad2 und 3 des TGF-ß/Activin Signalweg, als auch mit den Onkogenen Sno und Ski interagiert. Skip wirkt hier als Antagonist der Ski/Sno-Wirkung auf den TGF-ß/Activin-Signalweg und fungiert als Koaktivator (Leong et al., 2001). Die Interaktion zwischen Bx42 und der TGF-ß/Activin-Signalweg Komponente dSmad2, sowie mit dem Onkogen dSno konnte in dieser Arbeit auch für Drosophila bewiesen werden. Die Bedeutung dieser Wechselwirkung muss noch in weiteren Arbeiten analysiert werden. Der Einfluss der Bx42-RNAi-Induktion auf die TGF-ß/Dpp Zielgene distal-less (dll), optomotor blind (omb) und spalt (sal) wurde anhand von Reportergen Untersuchungen mit enhancer-trap-Linien und RNA in situ Hybridisierung untersucht. Es konnte gezeigt werden, dass das Ausschalten von Bx42 die Expression dieser Gene in ähnlicher Weise reprimiert, wie eine Elimination des TGF-ß/Dpp-Signals. Diese Ergebnisse unterstützen die Annahme, dass Bx42 in der Lage ist, TGF-ß/Dpp Zielgene durch eine Wechselwirkung mit Mad und Medea zu aktivieren. / The importance of Bx42 in Drosophila development was demonstrated using Bx42-RNA interference. The ubiquitous downregulation of Bx42 generated embryonic lethality, indicating the importance of this protein in early development. The tissue specific induction of Bx42-RNAi resulted in several different phenotypes depending on the driver line and the temperature at which animals were raised. The phenotypes obtained were the key point for the assumption that Bx42 may play a role in the regulation of a number of different cellular signalling pathways. Indeed, within the Notch signalling pathway Bx42 interacts genetically with Suppressor of hairless [Su(H)] and Notch intracellular domain (N-IC). Additionally, the reduction of Bx42 negatively affected the expression of the Notch target Genes cut (ct) and enhancer of split m8 [e(Spl)m8] (Negeri et al., 2002). In this work, the involvement of Bx42 in the Dpp signalling pathway was investigated. It was shown that Bx42 interacts both in vitro and in vivo, as demonstrated by yeast two hybrid protein-protein studies and Ni-NTA pull-down assays, with the TGF-ß/ Dpp components Mad and Medea. Domains of Smads (Mad and Medea) required for Bx42 interaction were examined using deletion constructs of Smads and the importance of the well conserved MH2 domains of Mad and Medea for this interaction was revealed. Moreover, the rescue of the Bx42-RNAi phenotype by the simultaneous overexpression of Medea demonstrated the genetic interaction between Bx42 and Medea. Furthermore, evidences for the interaction of Bx42 with the TGF-ß/Activin pathway component dSmad2 and with the oncogene protein dSno were obtained from interaction assays. The human homologue of Bx42, Skip, also interacts with Smad2/3 or Sno. The meaning of this interaction in Drosophila has yet to be analysed. The influence of Bx42-RNAi induction on the expression of Dpp target genes distal less (dll), optomotor blind (omb) and spalt (sal) was also investigated using enhancer trap lines and RNA in situ hybridisation. In this way it was proven that these genes are suppressed as they are by elimination of Dpp signalling. These results suggest that Bx42 may be able to modulate positively TGF-ß/Dpp signalling through an interaction with the signalling transducer Mad and Medea.

The amphibian species Xenopus laevis, along with mouse and chicken is a very important model system, used widely to dissect the molecular intricacies of various aspects of vertebrate development. Study with Xenopus has clear advantages in terms of various technical considerations including the ease of handling early stage of embryos and due to the remarkable documentation of several early molecular events during development. The concept of inductive interactions between various cell types during early development was first revealed by the studies performed in Xenopus, and among the various factors proposed for mesoderm induction, the members of transforming growth factor-β (TGF- β) superfamily have been considered to be the most probable candidates. About forty different members of the TGF-β superfamily have been cloned and characterized from various organisms. The superfamily members like activins and BMPs have been studied extensively with respect to their functional role during development. While BMPs were assigned as candidates for inducing ventral mesoderm, activins oppose the role of BMPs by inducing dorsal mesoderm. Studies that helped in delineating their roles were performed using three approaches that utilized the ligands, receptors or down stream signaling components (Smads). All the three components were studied with respect to their endogenous expression pattern and effects of ectopic expressions of the wild type or dominant negative mutants. These approaches led to the accumulation of evidences supporting the importance of these signaling molecules. All the above mentioned studies were only possible due to the cloning and characterization of cDNAs of the various proteins involved in the signaling pathway including the ligands. TGF-β2 and 5 are the two isoforms of TGF-β cloned from the amphibian system. We have earlier cloned and characterized the promoter for TGF-β5 gene, which suggested possible regulation of this factor by tissue specific transcription factors. Messenger RNA in situ hybridization analysis to study the TGF-β5-expression pattern during Xenopus development, showed spatial and temporal expression pattern. The expression was confined to specific regions that include notochord, somites, and tail bud among others, in the various stages analyzed. This suggested a possible role for TGF-β5 in organogenesis during the amphibian development. To better understand the role of TGF-β in Xenopus development, studies to examine the specific receptor expression pattern for this growth factor is very essential. With the lack of any reports on cloning of TGF-β receptors from this system, the aim of the present study was to isolate and characterize the receptors for TGF-β from Xenopus laevis. PCR cloning using degenerate primers based on the conserved kinase domains of this class of receptors, coupled to library screenings enabled the identification of two novel receptor cDNAs of the TGF-β receptor superfamily. Characterization of the isolated cDNAs suggested that one of them codes for a type II receptor for TGF-β. Further the cDNAs were found to be ubiquitously expressed during development, as judged by RT-PCR analysis. The cloned cDNAs can now be employed as tools, to study the expression pattern by means of mRNA in situ hybridization, on the various developmental stage embryos and to perform studies using antisense and dominant negative mRNA injection experiments in vivo. Such studies will greatly assist in delineating the role of TGF-β ligands and receptors during amphibian development.

[ES] La presente tesis doctoral titulada "New advanced anti-tumor therapies based on hybrid mesoporous nanodevices" se centra en el diseño, síntesis, caracterización y evaluación de nuevos nanodispositivos híbridos orgánico-inorgánicos. En concreto, se han desarrollado nanopartículas de sílice mesoporosas (MSNs) y nanopartículas Janus combinando MSNs con platino y con oro con el objetivo de mejorar los tratamientos anti-tumorales. El primer capítulo es una introducción general que incluye una visión global del contexto en el que se enmarca la investigación realizada. En particular, se incluye información básica sobre los diferentes tipos de nanopartículas empleados en esta tesis doctoral, así como la descripción de la enfermedad del cáncer y la aplicación de los nanomateriales como terapia. A continuación, en el segundo capítulo, se presentan los objetivos generales y específicos de esta tesis doctoral. Los capítulos tercero y cuarto describen dos estrategias terapéuticas basadas en el desarrollo de nanopartículas con movimiento para mejorar la terapia antitumoral. Concretamente, en el primer capítulo experimental se presenta un nanodispositivo autopropulsado para la liberación controlada de fármacos en respuesta al glutatión (GSH) intracelular. Éste se basa en nanopartículas tipo Janus compuestas por sílice mesoporosa y oro, funcionalizadas en la parte del oro con la enzima catalasa, cargadas con doxorrubicina y con cadenas de oligo(etilenglicol) (S-S-PEG) unidas por puentes disulfuro en la cara de la sílice. Una vez sintetizado y caracterizado el dispositivo, se confirmó su movimiento y se demostró el funcionamiento de la puerta molecular. Finalmente, la internalización celular y la liberación de doxorrubicina se estudiaron en cultivos celulares. Motivados por los resultados anteriores, en el cuarto capítulo se describe un nuevo nanomotor diseñado como tratamiento antitumoral y similar al anterior. En este caso, la nanopartícula Janus desarrollada está compuesta por una nanopartícula de sílice mesoporosa junto con una de platino, cargada con doxorrubicina y cubierta con S-S-PEG. Al igual que en el trabajo anterior, se consigue la autopropulsión del dispositivo. Además, se obtuvo el perfil cinético de liberación del cargo en respuesta a estímulos y se confirmó su aplicación en cultivos celulares. Los capítulos cinco y seis se centran en una estrategia terapéutica nueva que consiste en potenciar la acción del sistema inmune sobre el tumor para conseguir su eliminación. En el primero de estos capítulos experimentales, se utilizaron nanopartículas MSNs cargadas con el fármaco JQ-1 y un siRNA frente al factor de crecimiento transformante ßeta (TGF-ß) como inmunoterapia. Una vez sintetizadas y caracterizadas, se confirmó la capacidad de estas nanopartículas para llevar a cabo la liberación de los cargos, junto con la disminución en la expresión de PD-L1 y en la producción de TGF-ß. Por último, se confirmó su aplicabilidad al ser capaces de inducir la eliminación de células de melanoma por el sistema inmune. De acuerdo con el último capítulo experimental, se describe un nuevo enfoque inmunoterapéutico basado en la comunicación química. En este caso, empleamos una nanopartícula Janus de oro y sílice funcionalizada con un péptido llamado pHLIP en la cara de sílice y el anticuerpo contra el receptor PD-1 unido a la cara de oro (J-pHLIP-PD1). La membrana de la célula tumoral es decorada por este nanodispositivo y gracias a la exposición del anticuerpo PD-1, se consiguió atrapar a los linfocitos T circulantes, desencadenando la eliminación de células tumorales por el sistema inmunitario. Además, estos resultados se confirmaron en un modelo metastásico B16-F10-Luc con una reducción de nódulos metastáticos. Finalmente, en el capítulo séptimo y octavo, se aborda la discusión general y las conclusiones derivadas de los estudios experimentales presentados en esta tesis doctoral. / [CA] Aquesta tesi doctoral titulada "New advanced anti-tumor therapies based on hybrid mesoporous nanodevices" se centra en el disseny, síntesi, caracterització i avaluació de nous nanodispositius híbrids orgànic-inorgànics. En concret, s'han desenvolupat nanopartícules de sílice mesoporoses (MSNs) i nanopartícules Janus combinant MSNs amb platí i amb or per al tractament antitumoral. El primer capítol és una introducció general que inclou una visió global del context on s'emmarca la recerca realitzada. En particular, es presenta informació bàsica sobre les nanopartícules emprades en aquesta tesi doctoral, així com la descripció de la malaltia del càncer i l'aplicació dels nanomaterials com a teràpia. A continuació, al segon capítol, es presenten els objectius generals i específics d'aquesta tesi doctoral. Els capítols tercer i quart descriuen dues estratègies terapèutiques basades en el desenvolupmanet de nanopartícules amb moviment per aconseguir una millora de la terapia antitumoral. Concretament, al primer capítol experimental es presenta un nanodispositiu autopropulsat per a l'alliberament controlat de fàrmacs en resposta al glutatió (GSH) intracel·lular. Aquest es basa en nanopartícules tipus Janus compostes per sílice mesoporosa i or, funcionalitzades a la part de l'or amb l'enzim catalasa, carregades amb doxorrubicina i amb cadenes d'oligo(etilenglicol) (SS-PEG) unides per ponts disulfur a la cara de la sílice. Una vegada sintetitzat i caracteritzat el dispositiu, es va confirmar la seua capacitat de moviment i es va demostrar el funcionament correcte de la porta molecular. Finalment, la internalització cel·lular i l'alliberament de doxorrubicina es van estudiar en cultius cel·lulars. Motivats pels resultats anteriors, al quart capítol es descriu un nanomotor nou dissenyat com a tractament antitumoral i similar a l'anterior. En aquest cas, la nanopartícula Janus desenvolupada està composta per una nanopartícula de sílice mesoporosa juntament amb una de platí, carregada amb doxorrubicina i coberta amb S-S-PEG. Igual que en el treball anterior, s'aconsegueix autopropulsió del dispositiu. A més, es va obtenir el perfil cinètic d'alliberament del càrrec en resposta a estímuls i se'n va confirmar l'aplicació en cultius cel·lulars. Els capítols cinc i sis se centren en una estratègia terapèutica nova que consisteix a potenciar l'acció del sistema immune sobre el tumor per aconseguir-ne l'eliminació. Al primer d'aquests capítols experimentals, es van utilitzar nanopartícules MSNs carregades amb el fàrmac JQ-1 i un siRNA davant del factor de creixement transformant ßeta (TGF-ß) com a immunoteràpia. Un cop sintetitzades i caracteritzades, es va confirmar la capacitat d'aquestes nanopartícules per dur a terme l'alliberament dels càrrecs, juntament amb la disminució de l'expressió de PD-L1 i de la producció de TGF-ß. Finalment, se'n va confirmar l'aplicabilitat en ser capaços d'induir l'eliminació de cèl·lules de melanoma pel sistema immune. D'acord amb el darrer capítol experimental, es descriu un nou enfocament immunoterapèutic basat en la comunicació química. En aquest cas, fem servir una nanopartícula Janus d'or i sílice funcionalitzada amb un pèptid anomenat pHLIP a la cara de sílice i l'anticòs contra el receptor PD-1 unit a la cara d'or (J-pHLIP-PD1). La membrana de la cèl·lula tumoral és decorada per aquest nanodispositiu i gràcies a l'exposició de l'anticòs PD-1, es va aconseguir atrapar els limfòcits T circulants, desencadenant l'eliminació de cèl·lules tumorals pel sistema immunitari. A més, aquests resultats es van confirmar en un model metastàtic B16-F10-Luc amb una reducció de nòduls metastàtics. Finalment, al capítol setè i vuitè, s'aborda la discussió general i les conclusions derivades dels estudis experimentals presentats en aquesta tesi doctoral. / [EN] The present PhD thesis entitled "New advanced anti-tumor therapies based on hybrid mesoporous nanodevices" focuses on the design, synthesis, characterization, and evaluation of new hybrid organic-inorganic nanodevices. We have developed mesoporous silica nanoparticles (MSNs) and Janus platinum-MSN and gold-MSN nanoparticles for tumor treatment. The first chapter is a general introduction that includes an overview of the context related to the research developed in this thesis. In particular, it includes basic information about different nanoparticles used in this doctoral thesis along with the description of cancer disease characteristics and the application of nanomaterials as therapy. Next, in the second chapter, the general and specific objectives of this Ph.D. thesis are presented. The third and fourth chapters describe two nanotechnology-based therapeutic strategies based on the development of nanomotors to improve cancer therapy. Specifically, the first experimental chapter presents a self-moving nanodevice for controlled drug release in response to intracellular glutathione (GSH). It is based on Janus gold-mesoporous silica nanoparticles functionalized with the enzyme catalase in the gold face, loaded with doxorubicin and capped with disulfide-linked oligo(ethylene glycol) (S-S-PEG) chains on the silica face. Once synthesized and characterized, the nanosystem motion was confirmed and the proper gating mechanism of the nanodevice was proven. Finally, the cellular uptake and doxorubicin release capacity have been demonstrated in cell cultures. Encouraged by the above results, chapter four describes a similar nanomotor design for antitumor therapy. In this case, the nanoparticle developed is composed of a Janus platinum-mesoporous silica nanoparticle, loaded with doxorubicin, and capped with S-S-PEG. As well as in the previous work, self-propulsion of the nanoparticles was achieved. Moreover, the stimuli-responsive cargo release kinetic profile was obtained and its application was confirmed in cell cultures. Chapters five and six focus on a new therapeutic strategy, empowering the immune system action on tumors to reach tumor cell death. In the first of these experimental chapters, JQ-1 and transforming growth factor-beta (TGF-ß) siRNA-loaded nanoparticles were used as efficient tumor immunotherapy. Once synthesized and characterized, the efficient cargo delivery was accomplished along with the programmed death-ligand 1 (PD-L1) downregulation and TGF-ß silencing. Lastly, its application was confirmed by triggering a specific immunogenic removal of tumor cells in melanoma cells. In chapter six, the development of a new communication-based immunotherapeutic approach is reported. In this case, we employ Janus gold-MSN functionalized with a peptide called pHLIP onto silica face and anti-PD-1 antibody bound to gold face (J-pHLIP-PD1). Tumor cell membrane is decorated by this nanodevice, leaving exposed on the surface PD-1 antibody which catches circulating T lymphocytes. It triggers immune system-induced-tumor leveling. Moreover, J-pHLIP-PD1 treatment-associated reduction of metastatic burden was also proven. Finally, in the seventh and eighth chapter, the general discussion and conclusions derived from the presented experimental studies of this Ph.D. thesis are exposed. / The authors thank the Spanish Government (Projects MAT2015-64139-C4-1, AGL2015-70235-C2-2-R, CTQ2014-58989-P, CTQ2015-71936-REDT and CTQ2017-87954-P) and the Generalitat Valencia (PROMETEO/2018/024) for support. The Comunidad de Madrid (IND2017/BMD-7642) is also gratefully acknowledged. The authors thank the Spanish Government (project PID2021-126304OB-C41, (MCUI/FEDER, EU)) and the Generalitat Valenciana (project PROMETEO CIPROM/2021/007). E.L-S is grateful for her FPU fellowship funded by MINECO (FPU18/06539). This work was supported by the European Research Council (ERC) via Advanced Grant (101052997, EDISON). This study forms part of the Advanced Materials program (MFA/2022/049) and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana. This work was supported by the European Research Council (ERC) via Advanced Grant (101052997, EDISON) and by CIBER -Consorcio Centro de Investigación Biomédica en Red- (CB06/01/2012), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación. / Lucena Sánchez, E. (2024). New advanced anti-tumor therapies based on hybrid mesoporous nanodevices [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/205528