Le cancer du sein est une maladie impactant le système immunitaire dont le rôle évolue au cours de la tumorigénèse, allant de la détection et l'élimination des cellules transformées (immunosurveillance) à la promotion du développement tumoral (immunosubversion). Les efforts déployés pour définir de nouvelles stratégies thérapeutiques ont révélé que rétablir l'immunité anti-tumorale chez les patientes permettrait d'améliorer leur pronostic. Durant ma thèse, nous avons mis en évidence l'existence de signaux activateurs et inhibateurs des pDC dans les cancers du sein, qui confèrent aux pDC un rôle dans l'immunosurveillance et dans l'échappement immunitaire du cancer du sein respectivement. Nous avons ainsi montré que le TGF-beta et le TNF-alpha sont impliqués dans l'inhibition fonctionnelle des TApDC en réprimant l'expression et l'activation d'IRF-7. Dans un second temps, nous avons montré i) la présence de complexes [ADN-LL37] produits par les neutrophiles dans les tumeurs et capables d'induire la production d'IFN-alpha par les pDC, ii) l'expression des gènes associés aux IFN-I dans les tumeurs de sein et iii) un rôle majeur de la voie des IFN-I dans l'immunosurveillance des tumeurs mammaires chez la souris. De plus, des données préliminaires chez la souris suggèrent que les pDC participent à l'immunosurveillance anti-tumorale in vivo. Les travaux présentés dans ce manuscrit apportent de nouvelles données sur le rôle des pDC dans l'immunosurveillance des cancers du sein et ouvrent sur de nouvelles stratégies d'immunothérapie anti-tumorale ciblant les pDC / Breast cancer are disease impacting immune system whose play role during tumorigenesis, to detect and eliminate malign cells (immunosurveillance) or promote tumoral development (immunosubversion). Efforts to define new therapeutic strategies revealed that restoring anti-tumor immunity in patients would improve their prognosis. During my thesis, first, we demonstrated the existence of stimulatory and inhibitory signals of pDCs in the breast, which give the pDCs a role in immunosurveillance and immune escape of breast cancer, respectively. We showed that TGF-beta and TNF-alpha are involved in the functional inhibition of TApDC repressing IRF-7 expression and activation. Secondly, we showed i) the presence of [DNA LL37] complex produced by neutrophils in tumors that can induce the production of IFN-alpha by pDCs, ii) the expression of type I IFN associated genes in breast tumors and iii) a major role of IFN-I pathway in immunosurveillance of mammary tumors in mice. In addition, in mice, preliminary data suggest that pDC could play a role in anti-tumor immunosurveillance in vivo. The work presented in this thesis provide new data on the role of pDCs in immunosurveillance of breast cancers, and open new anti-tumor immunotherapy strategies targeting pDCs

Arkadia participa do processo de amplificação da sinalização de TGF-b mediada por Smads, via degradação do I-Smad. O objetivo desse estudo foi caracterizar e investigar a influência de Arkadia em linhagens celulares de cânceres de tiróide. A expressão gênica de Arkadia em linhagens celulares de carcinomas papílifero (NPA), folicular (WRO) e anaplásico (ARO), foi avaliada por PCR quantitativo. Em ARO, que apresenta a maior expressão de Arkadia, foram identificados subclones (ARO_1 e ARO_2) com expressão diferencial de Arkadia, ARO_2>ARO_1. A expressão gênica de SMAD2, 3, 4, 7 e de genes do ciclo celular modulados por TGF-b, foi maior em ARO_2. Os subclones respondem ao tratamento com peptídeo de TGF-b1 e activina A. O crescimento in vivo (xenotransplante) mostra que ARO_2 desenvolve um tumor de menor volume. Recentemente a origem de ARO foi questionada e comprovamos sua origem por análises de expressão gênica e morfologias. Desta maneira, observamos que a expressão diferencial de Arkadia indica que ela está envolvida na modulação inibitória da via de TGF-b. / Arkadia is involved in the process of amplification of the TGF-b signaling mediated by Smads, by degradation of I-Smad. The aim of this study was to characterize and investigate the influence of Arkadia in thyroid cancers cell lines. Arkadia gene expression in the papillary (NPA), follicular (WRO) and anaplastic carcinoma cell lines (ARO) was evaluated by quantitative PCR. In ARO, which presents the highest Arkadia expression, we identified subclones (ARO_1 and ARO_2) with differential Arkadia expression ARO_2> ARO_1. The expression of SMAD2, 3, 4, 7 and the cell cycle genes modulated by TGF-b, was also higher in ARO_2. However both the subclones responded to treatment with peptide of TGF-b1 and activin A. The in vivo growth (evaluated by xenotransplant), showed that ARO_2 developed tumors of lower volume. Recently the ARO origin was questioned and we proved its origin by gene expression and morphological analysis. This way, the differential Arkadia expression indicates that it is involved in modulation of the inhibitory TGF-b pathway.

Zirkadiane Uhren sind Zell-autonome Oszillatoren. Aus diesem Grund ist deren interzelluläre Kopplung essentiell, um die Synchronität zirkadianer Oszillatornetzwerke zu erhalten und die Störung zirkadianer Gewebsfunktionen zu verhindern. Neuronale Oszillatoren des Nucleus suprachiasmaticus (SCN), der Schrittmacher-Uhr im Zentralnervensystem der Säugetiere, koppeln interzellulär und über den Austausch sekretierter Neurotransmitter. Die Fähigkeit zirkadianen Oszillatoren peripherer Gewebe interzellulär zu koppeln ist hingegen stark umstritten und molekulare Mechanismen sind unbekannt. In dieser Dissertation zeigen wir, dass periphere Oszillatoren in der Tat interzellulär über den Austausch sekretierter Signalmoleküle koppeln und identifizieren TGF-b als peripheren Kopplungsfaktor. Weiterhin zeigen wir, dass TGF-b die cAMP Enhancer-Motiv abhängige, als auch Immediate Early Expression des Uhr-Gens PER2 induziert und folglich die Phasenanpassung molekularer zirkadianer Oszillationen reguliert. Genetische und pharmakologische Störeinflüsse verursachen die Dysregulation des TGF-b Signalweges und begünstigen die Desynchronisierung zellulärer Oszillatoren, welche sich in Amplitudenreduktion und verstärkter Sensitivität gegenüber Zeitgeber-Signalen äußert. Die in dieser Dissertation präsentierten Ergebnisse, legen einen bisher unbekannten molekularen Mechanismus intrazellulärer Kopplung peripherer zirkadianer Oszillatoren dar und eröffnen neue Perspektiven auf die Bedeutung der Synchronität peripher zirkadianer Uhren für rhythmische Organfunktionen und zirkadiane Gesundheit. / Circadian clocks are cell-autonomous oscillators. Intercellular coupling is crucial to prevent desynchronization of oscillator networks and thus, the disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus (SCN), couple intercellularly via the exchange of secreted neurotransmitters, intercellular coupling among peripheral oscillators is highly debated and molecular mechanisms are unknown. Here, we show that peripheral circadian oscillators couple intercellularly via exchange of secreted signaling molecules and identify TGF-ß as peripheral coupling factor. TGF-ß signaling induces the cAMP response element dependent, immediate-early expression of the clock gene PER2, thereby phase-adjusting the molecular circadian oscillator. Genetic or pharmacologic disruption of TGF-ß signaling causes desynchronization of cellular oscillators resulting in amplitude reduction and increased sensitivity towards Zeitgeber cues. Our findings reveal a previously unknown mechanism of peripheral coupling and open new perspectives on the importance of peripheral clock synchrony for rhythmic organ functions and circadian health.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / ADAMs (a disintegrin and metalloprotease) can influence multiple cellular processes involved in normal development and pathogenesis. ADAM12 expression levels are elevated in many pathological conditions including cancer, cardiovascular disease, and muscle regeneration. Recently, ADAM12 has emerged as a candidate cancer gene in a comprehensive genetic analysis of human breast cancers. The regulation of ADAM12 expression is poorly understood. Identification of new substrates for ADAM12 metalloprotease can expand our knowledge of processes in which ADAM12 is involved. Here, we show that ADAM12 expression is upregulated by transforming growth factor beta (TGF-beta), an essential signaling pathway for many cellular processes. This upregulation requires proteosomal degradation of a transcriptional repressor SnoN. Furthermore, breast cancer cell lines expressing high levels of SnoN have significantly impaired induction of ADAM12 by TGF-beta, suggesting an inverse correlation between SnoN and the extent of regulation of ADAM12 by TGF-beta. Additionally, we demonstrate that ADAM12 is one of the metalloproteases involved in shedding a Notch ligand, Delta like 1 (Dll1). The Notch signaling pathway plays a crucial role in cell fate decision during development and in adults. Cleavage of Dll1 by ADAMs occurs in cis and results in activation of Notch signaling in a cell-autonomous manner. Furthermore, the intracellular domain of Dll1 created after cleavage further enhances TGF-beta signaling in response to TGF-beta. Our analysis of breast cancer-associated mutations in the ADAM12 gene showed a lack of proper proteolytic processing of the ADAM12 protein and its mislocalization to the endoplasmic reticulum. Additionally, ADAM12 mutants show a dominant-negative effect on the processing of the wild-type ADAM12 and result in loss of the functional ADAM12 at the cell surface. Collectively, our results indicate a new mechanism of regulation of ADAM12 expression, expand the role of ADAM12 in the regulation of Notch signaling, and characterize cancer-associated mutations in the ADAM12 gene.

TGFβ is an immunoregulatory cytokine that has a pivotal function in maintenance of immune tolerance via the control of lymphocyte proliferation, differentiation and survival. Defects in TGFβ1 expression or in its signaling in T cells correlate with the onset of several autoimmune diseases. However, the early effects of this cytokine on the innate immune system, particularly the dendritic cells (DCs) which play an equally important role in development of immune tolerance, are not well documented in vivo. In the current study, we developed conditional knockout mice with targeted deletion of Tgfbr2 specifically in dendritic cells. DC-Tgfbr2 KO mice developed spontaneous multi-organ autoimmune inflammation with T and B cell activation. Phenotypic analysis of dendritic cells revealed no significant differences in the expression of MHCII and co-stimulatory molecules between control and DC-Tgfbr2 KO mice. However, we found that DCs from DC-Tgfbr2 KO mice were more pro-inflammatory, which exacerbated the severity of disease in a T cell transfer model of colitis. Furthermore, increased IFNγ expression by Tgfbr2-deficient DCs inhibited antigen-specific regulatory T cells (Tregs) differentiation by DCs in the presence of TGFβ. Since DCs play an important role in Treg homeostasis in vivo, we also examined the phenotype of Tregs and observed a significant increase in the frequency and numbers of Foxp3⁺ T cells in both the spleen and MLNs of DC- Tgfbr2 KO mice. Further analysis of these Tregs revealed attenuated expression of Foxp3 and an expansion in the numbers of CD4⁺CD25⁻Foxp3⁺T cells suggesting that the Tregs from KO mice may not be fully immunosuppressive. Adoptive transfer of in vitro differentiated iTregs into 2-3 week old DC-Tgfbr2 KO mice partially rescued the autoimmune phenotype by reducing the frequency of activated T cells and severity of colitis but did not prevent inflammation in other organs. The phenotype of this novel mouse model clearly indicates the importance of TGFβ signaling in DCs in the maintenance of immune homeostasis and prevention of autoimmunity and provides an opportunity to study the pathogenesis of complex disorders such as autoimmune gastritis, pancreatitis, hepatitis and inflammatory bowel diseases.

Long interspersed nuclear element-1 (L1) is a genetic element that mobilizes throughout the mammalian genome via retrotransposition and damages host DNA via mutational insertions, chromosomal rearrangements, and reprogramming of gene expression. The cellular mechanisms responsible for aberrant L1 expression during cancer pathogenesis are unclear. Previously, we have shown that L1 reactivation in several human cell lines is dependent upon the activation of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor member of the PAS superfamily of proteins. We also showed that ectopic expression of L1 reprograms the HepG2 genome leading to epithelial-to-mesenchymal transition (EMT). Here we present evidence that reactivation of L1 and modulation of EMT in HepG2 cells by the AhR ligand benzo(a)pyrene (BaP) is effected through the canonical TGF-β1 signaling pathway. BaP increased TGF-β1 mRNA, SMAD2 phosphorylation and decreased expression of E-Cadherin. The functional relevance of these interactions and the involvement of TGFBR1/ALK5 and SMAD2/3 were confirmed by siRNA interference. Furthermore, expression of L1-encoded ORF1p was positively correlated with the activation of TGF-β1 signaling in human hepatocarcinoma samples at various stages of malignant progression. These results indicate that ligand-mediated AhR activation regulates L1 via canonical TGF-β1 signaling and raise important questions about the molecular etiology of human hepatocarcinomas.

The worm Caenorhabditis elegans is a well-studied model organism in numerous aspects of its biology. This small free living nematode has less than 1,000 cells, but shows clear conservation in both signaling and behavior to mammals in aspects of appetite control. This is of importance to humans, where failure of appetite control is a major factor in the unprecedented obesity epidemic that we see today. In general, worm behavior reflects its internal nutritional state and the availability and quality of food. Specifically, worms show a behavioral state that mimics aspects of the mammalian behavioral satiety sequence, which has been termed satiety quiescence. We have used locomotion tracking and Hidden Markov Model analysis to identify worm behavioral state over time, finding quiescence along with the established worm locomotive behaviors roaming and dwelling. Using this analysis as well as more conventional cell biology and genetic approaches we have further investigated satiety signaling pathways. We have found that the neuron ASI is a major center of integration of signals regarding the internal nutritional state of the worms as well as the nutritional content of its environment. Our results show that cGMP causes levels of the TGFβ ligand to be increased in fasted worms, which is then released and binds to its receptor on the RIM and RIC neurons. This signaling connects nutritional state to behavioral response, promoting the sleep-like behavioral state satiety quiescence. Additionally, we have begun a candidate approach examining several other groups of signaling molecules for potential roles in satiety quiescence signaling including cannabinoids, multidrug resistance proteins, and neuropeptides. The result of this investigation is a better understanding of mechanisms of satiety quiescence signaling as well as a new tool that provides highly quantitative, unbiased, and automated data to aid in our ongoing work.

151 8 SUMMARY Recombinant vaccinia virus has been used for elicitation of the immune response against expressed heterologous proteins which has led to protection of the host organisms against the agents producing that antigen (viruses, cancer cells). In our laboratory, we designed and evaluated several vaccines against cancer caused by human papillomavirus type 16 (HPV16). Vaccinia viruses derived from replication competent strain P13 or attenuated MVA were used for construction of recombinant viruses expressing HPV16-E7 in highly immunogenic fusion construct SigE7LAMP. Recombinant viruses were used both in prophylactic and therapeutic settings in mouse tumor models using TC-1 or TC-1/A9 cells. The genes encoding stimulatory cytokines GM-CSF or Flt3 ligand were inserted into the above viruses to support the immune system and to potentiate the anticancer response. Tumor microenvironment was modified using the recombinant viruses expressing both the E7 gene and soluble receptor for TGF-β which should decrease the inhibition of immune system caused by tumor TGF-β cytokine and elicit the response against tumor cells. Intratumoral or intraperitoneal administration of viruses enhanced anticancer response in mice, the viruses expressing Flt3 ligand induced the proliferation of E7- specific cytotoxic T lymphocytes....

Este trabalho avaliou o efeito da dieta hiperlipídica em camundongos para o estudo da inflamação cardiovascular. Camundongos C57Bl/6 machos com 8 semanas de vida foram utilizados nos ensaios, sendo divididos nos grupos dieta controle e dieta hiperlipídica. Após 8 semanas foram avaliados: o ganho de peso, a porcentagem de tecido adiposo, pressão arterial sistólica, frequência cardíaca, perfil lipídico e glicêmico séricos. A partir de cortes histológicos de aortas e corações corados com picrossirius foram feitas análises morfométricas. Em cortes histológicos de aorta foram realizadas a análise fibras elásticas e colágenas usando a coloração de Weigert-Van Gieson. Também foram realizadas a quantificação de fibras colágenas em aortas, usando a coloração de picrossirius. Nos tecidos aórticos e cardíacos foram feitos: 1) Ensaios de atividade enzimática para ECA e MPO. 2) Ensaios de Immunoblotting para a detecção proteíca para ECA e TGF-b. Também foram feitos ensaios de imuno-histoquímica para marcação e localização de ECA e TGF-b no tecido aórtico. / This work evaluated the effect of high-fat diet in mice for the study of cardiovascular inflammation. C57BL / 6 mice at 8 weeks of age were used in the tests were divided in groups control diet and high fat diet. After 8 weeks were evaluated: weight gain, percentage of fat, systolic blood pressure, heart rate, serum lipids and glucose levels. From histological aortas and hearts stained with picrosirius morphometric analyzes. Histological sections of the aorta were performed to analyze elastic and collagen fibers using Weigert-Van Gieson staining. Also the quantification of collagen fibers in aortas using picrosirius staining. In aortic and cardiac tissues were made: 1) Enzymatic activity assays for ACE and MPO. 2) Immunoblotting assays to detect proteinous for ACE and TGF-b. Also were peformed Immunohistochemistry assays for marking and localization of ACE and TGF- b in the aortic tissue.

Blood vessels are dynamic structures whose properties are continuously adapted by resident vascular cells. Existing mechanobiological models tend to ignore regulatory signalling and cell population dynamics, both key determinants of arterial growth and remodelling (G&R). In this D.Phil., a combined theoretical, experimental and computational approach is used to formulate, refine and implement a novel model of the arterial wall that includes vascular mechanics, microstructure, biochemical metabolism and signalling, and cell phenotype and population dynamics. A mathematical chemo-mechano-biological (CMB) model is formulated by coupling a biomechanical model of the arterial wall as a cylindrical nonlinear elastic membrane to a system of biologically-informed evolution laws governing fibroblast cell-mediated, transforming growth factor (TGF)-β-regulated collagen metabolism. Model simulation of inflammatory aneurysm development suggests that increasing TGF-β levels promotes a cell-driven profibrotic response leading to aneurysm stabilisation, illustrating the model's ability to couple chemo-biological processes to tissue-level mechanical evolution. To inform the theoretical framework experimentally, a recent mouse model of post-developmental disruption of medial smooth muscle TGF-β signalling is for the first time subjected to hypertension, and characterised by biaxial mechanical testing and (immuno)histological staining. Increased adventitial TGF-β levels following perturbation are associated with strong profibrotic responses (increased cellularity, collagen deposition, thicker walls) altering tissue mechanics (lower biaxial stress, higher structural stiffness). Simulation of realistic arterial geometries is enabled by coupling the 1D CMB model to a three-dimensional structural solver. Heterogeneous spatial distributions of mechanical, microstructural and chemo-biological variables determining the evolution of complex saccular aneurysm geometries can be simulated with this 3D implementation. A novel chemo-mechano-biological model of vascular cell dynamics and regulatory signalling governing arterial G&R is formulated, informed by specifically-generated experimental data, and implemented in an advanced 3D computational framework. This will allow for virtual investigation of therapies acting on chemo-biological agents of arterial G&R, with potential benefits for vascular disease patients.