Дисертації з теми "Notch pathway signalling"

The Notch signalling pathway is important in development and differentiation of a diverse range of both embryonic and adult tissues. There is now strong evidence implicating aberrant Notch signalling in the pathogenesis of T-cell acute lymphoblastic leukaemia (T-ALL), with over 50% of paediatric patients having activating mutations in NOTCH-1. This thesis aims to explore several aspects of the Notch pathway in both T-ALL and acute myeloid leukaemia (AML). Chapter one summarises the published data on the Notch signalling pathway itself, addressing the basic understanding of Notch activation through cell-to-cell interaction, as well as the mechanisms through which it is regulated. The role that Notch signaling plays in normal haematopoiesis is also discussed. Chapter three addresses the incidence and characteristics of NOTCH-1 mutations in a cohort of adult patients with T-ALL in comparison to the published study of paediatric T-ALL, as well as in a cohort of patients with infantile leukaemia and AML. Secondly, the prognostic significance of NOTCH-1 and FBXW7 mutation status of adult T-ALL patients treated on the UKALLXII was investigated. Thirdly, a novel mutation affecting the LNR domain of NOTCH-1 is reported. Chapter four includes data quantifying NOTCH-1 mutation level in T-ALL patients, as well as the stability of NOTCH-1 mutations at presentation and relapse. These data indicate NOTCH-1 mutations commonly occur as secondary events in leukaemia pathogenesis, and suggest widespread clonal heterogeneity in T-ALL. Chapter five explores the functional and prognostic consequences of a novel alternatively spliced isoform of the CSL transcription factor in AML, which was termed CSL-TREX (for TRuncates Exon X). The functional consequences of expressing CSL-TREX in CD34+ cells, in luciferase reporter assays and co-immunoprecipitation experiments with NOTCH-1 is reported. Chapter six summarises the overall implications of these findings to T-ALL and AML, and the future directions of research in this area.

This project contributes to our understanding of bone cell biology and sheds light on the potential therapeutic application of Notch signalling pathway on bone-related diseases. The thesis was a step forward in answering how bone cells communicate with each other and determinate their own fates. It provides the first evidence demonstrating Notch signalling is critical in bone cell functions.

Haematopoiesis is the process to produce haematopoietic stem cells (HSCs), haematopoietic progenitors (HPCs) and terminally differentiated cell types. In the adult, HSCs resided in bone marrow while in the embryo, haematopoiesis occurred sequentially in several niches including yolk sac, aorta-gonad-mesonephros (AGM) region, placenta and fetal liver. The AGM region is the first place where HSCs arise in vivo and therefore should provide important factors to induce haematopoiesis. The mouse embryonic stem cells (mESC) system is a powerful platform to mimic the development process in vitro and is widely utilized to study the underlying mechanisms because they are pluripotent and can be genetically manipulated. A novel co-culture system has been established by culturing differentiating mESCs with primary E10.5 AGM explants and a panel of clonal stromal cell lines derived from dorsal aorta and surrounding mesenchyme (AM) in AGM region. Results of these co-culture studies suggested that the AM-derived stromal cell lines could be a potent resource of signals to enhance haematopoiesis. Molecular mechanism involved in haematopoiesis is a key research direction for understanding the regulation network of haematopoiesis and for further clinical research. A series of studies have demonstrated involvement of the Notch signalling pathway in haematopoiesis during development but with controversial conclusions because of the difference of models concerning various time windows and manipulating populations. This project aimed to investigate the role of Notch signalling pathway during haematopoiesis in the AGM environment. We analyzed the expression of Notch ligands in AGM-derived stromal cells with or without haematopoietic enhancing ability. No correlation was observed between ligand expression and haematopoietic enhancing ability in stromal cell lines or between Notch activity in EBs and haematopoietic enhancing ability. We demonstrated that inhibition of the Notch signalling pathway using the γ-secretase inhibitor could abrogate Notch activity in both mES-derived cells and the haematopoietic enhancing AM stromal cell line. To better understand the involvement of the Notch signalling pathway in a more specific spatial-temporal environment, we established a co-culture system of haemangioblast like cells (Flk1+) with one of AM region derived stromal cell lines with haematopoietic enhancing ability . We found that the AM stromal cell line could enhance Flk1+ derived haematopoiesis as assessed by haematopoietic colony formation activity and production of CD41+cKit+ progenitor cells. Based on the issue that the inhibitor could potentially affect both the ES cells and stromal cells, we carried out genetic approaches to overexpress or knock down Notch signalling pathway in this Flk1+/AM co-culture system. Interestingly, it was found that when Notch activity was enhanced in Flk1+ cells, the production of haematopoietic progenitors was inhibited and the number of cells expressing the pan-haematopoietic marker CD45 was reduced. By using the inducible dominant negative MAML1 system to knock down Notch activity, it was found that the haematopoiesis in the Flk1+/AM co-culture system was not affected, which could be accounted for the low Notch activity in this system. These results supported the hypothesis that the Notch signalling pathway plays a role in modulating Flk1+ derived haematopoietic differentiation within the AGM microenvironment.

The Notch receptor is part of a highly conserved omnipresent developmental pathway that has crucial roles in developing and self-renewing tissues. During activation of the signalling pathway Notch binds to its ligand, presented on a neighbouring cell. It is thought that this results in a conformational change within the Negative Regulatory Region (NRR) unmasking a key proteolytic site (S2) and allows for metalloprotease cleavage. This facilitates further cleavage by gamma-secretase, initiating downstream events. Thus far, the molecular mechanism by which the S2 site is revealed has not been defined, though indirect evidence favours a model whereby transendocytosis of the Notch extracellular domain into the ligand bearing cell results in mechanical unfolding of the NRR. Research presented here suggests the NRR of human Notch2 (hN2) unfolds within a mechanosensing force range. Furthermore, through the application of a force (200 pN) the hN2-NRR was shown to unfold sufficiently to expose the S2 site allowing cleavage by metalloproteases. Molecular dynamics (MD) simulations offer insight into the unfolding process of the hN2-NRR, revealing near-sequential unfolding of its constituent LNR and HD domains. Removing the linker region between LNR’s A and B appears to be the first force ‘barrier’ in the unfolding pathway, producing the largest increase in solvent accessibility at the S2 cleavage site. Through docking simulations, this unfolding event was shown to expose the S2 cleavage site sufficiently to allow access to the metalloprotease TACE. Removing coordinated metal ions from the hN2-NRR structure resulted in a dramatic decrease in the forces required for unfolding during AFM experiments, highlighting their role in increasing the resistance of the hN2-NRR to forced unfolding. Removal of disulphide bonds within the structure resulted in a loss of detectable LNR unfolding, highlighting their role in LNR stabilisation.Six HD destabilising mutants, characterised through their role in the hN1 disease, T-cell Acute Lymphoblastic Leukemia, showed three key changes to the unfolding pathway of the hN2-NRR. Firstly, mutants A1647P, L1573P and V1623D showed a dramatic decrease in force required for unfolding in AFM experiments. MD simulations highlighted a lack of force required for the unfolding the LNRA:B linker previously characterised as the key event in removing NRR autoinhibition. Secondly, all the mutants studied here showed changes to the stability of the alpha3-helix (within the HD domain) resulting in transient shifts or bending during unfolding of the LNRA:B linker and the LNRB. Finally, changes were observed within the LNRC of A1647P and L1566P. Within these mutants the LNRC was observed to be unfolding, an event not present during wild-type unfolding. Within mutant L1566P this is thought to be due to the disruption of the conserved salt-bridge occurring between Arg1567 (HD domain) and Asp1506 (LNRC). Within mutant A1647P this is likely due to widespread domain destabilisation. Overall, research presented here has provided the first direct evidence that the NRR is mechanosensing and that mechanical force can allow for cleavage at the S2 site. Further characterisation has been performed to analyse the unfolding pathway through ion chelation, disulphide oxidation and mutagenesis studies.

Thymic epithelial cells (TECs) are indispensable for the development of T cells in the thymus. Two subtypes of TECs exist in the thymus, medullary mTECs and cortical cTECs. Both mTECs and cTECs originate from endodermal thymic epithelial progenitor cells (TEPCs) in the embryo, but how the differentiation of TEPCs is regulated is not well understood. The aims of this thesis were to establish the role of Notch signalling in TEPC differentiation, and how it interacts with known regulators such as FOXN1 and the NFκB pathway. Gene expression data showed that Notch is active in TEPCs and exhibits a correlation with the mTEC lineage. Loss of Notch function led to a significant reduction in the number of mTECs in the thymus, and this can be attributed to aberrant mTEC specification. Furthermore, the duration of Notch activity in determining mTEC number appears limited to the early phase of organogenesis, and precedes RANK/NFκB mediated mTEC proliferation. Gain of Notch function resulted in a considerable shift to a primitive, TEPC-like phenotype, and subsequently a latent increase in mTEC frequency. Finally, transcriptomic and functional analyses pointed to a cross-repressive mechanism between Notch and FOXN1 in TEPCs. Taken together, these results identified Notch as a novel regulator of mTEC specification, likely through maintaining the potency of fetal TEPCs, a prerequisite for mTEC lineage commitment.

The cell surface Notch receptor and its ligand, presented on adjacent cells, bind through their extracellular regions and lead to the release of Notch intracellular domain (NICD). NICD then translocates to the nucleus to form the Notch transcription activating complex. ANK domain of NICD plays an essential part in the formation of this complex and the role of ANK domain was analysed both structurally and functionally using site directed mutagenesis. A single residue E2072 on the sixth ANK repeat was found crucial to maintain the stability of the Notch transcription activating complex. It can therefore be considered as a putative “hot-spot” for the purpose of drug discovery. Recently the interaction of Neutralized (an E3 ubiquitin ligase) with Tom (a Bearded protein) was identified. This study was undertaken to understand and structurally characterise the Tom-Neuralized interaction. Expression constructs were designed using the information from sequence analysis of Tom and Neuralized proteins and expressed using bacterial expression system. The NHR1 domain, expressed as an N-terminal His-tag protein, was used to determine the crystal structure of NHR1 domain. The structure was found to be structurally related to SPRY proteins consisting of jelly roll topology. Potential binding sites were predicted that localised on a cluster referred as ‘hydrophobic patch’ in this study. The expression of Tom remained insoluble under most experimental conditions and therefore a 12 amino acid peptide was synthesized. Biophysical analysis showed the binding affinity between NHR1 and Tom was weak but still could form the complex. Comparison of NHR and SPRY domain proteins suggested that the binding interface of the two could be common indicating that the interaction of Delta could require the same binding interface of NHR1 domain as Tom. Lastly, interaction of NHR1 with PI4P was analysed and verified using interfacial studies, which showed that NHR1 protein localised below the lipidic layer.

In the mid-gestation embryo, the first definitive transplantable hematopoietic stem cells (dHSCs) emerge by embryonic day E10.5-E11 in the aorta-gonadomesonephros (AGM) region, as a result of a step-wise maturation of precursors called pre-HSCs. The analysis of several Notch mutants suggests that Notch signalling is essential for the execution of the definitive hematopoietic programme in the AGM. Mouse embryos deficient for Notch1, RBP-Jk or Jagged1 cannot efficiently generate intra-embryonic hematopoeitic progenitors. It has also been reported that knockdown of Notch target genes (Hes1, Hes5) results in hematopoietic impairment. However a clear picture of the role of Notch pathway in HSC development is still missing. In this work we characterised precise stages and cell types during HSC development in which Notch signalling is involved. First we used a Hes1-dEGFP reporter mouse line that allowed us to monitor Notch pathway activity in a narrow window of time. The results suggest that the level of Notch activity fluctuates in HSC lineage in the AGM region and is down-regulated in dHSCs in the foetal liver (where dHSCs migrate after generation in the AGM region). By using transplantation assay, we further showed that fluctuations of Notch activity are essential for HSC development, and that this pattern in the HSC lineage might work as a switch between maturation and proliferation of PreHSC1, PreHSC2 and dHSC, in which temporary decrease might be required to mature from one type to another, both in vitro and in vivo. These findings might need to be taken into consideration for in vitro generation of haematopoietic stem cells, where a fine tuning of Notch signalling activity could greatly improve their emergence.

Wool follicle development is initiated during foetal life through signals exchanged between the mesenchyme and epithelium. Follicle primordia are initially visible as condensations of dermal prepapilla cells, and are associated with epithelial thickenings. The dermal papilla is believed to secrete signalling molecules that direct the rate of cell division in surrounding bulb cells in the mature wool follicle. This determines the amount of fibre grown. It has been hypothesised that the dermal papilla cell population of the skin is finite and is determined at follicle initiation in the foetus. It is thought that this population defines the total quantity of fibre producing tissue in the mature sheep. The developmental mechanisms that specify this cell population and control their distribution in the growing follicle population is not known. There is however, evidence implicating the Notch signalling pathway in foetal skin development. Some Notch signalling pathway genes have been identified in ovine skin and other tissue, including the incomplete sequences of Notch-1, Jagged-1, Delta—1 and Delta-4. This study reports on the roles of some key Notch genes prior to (d 56) and during (d 70) primary follicle initiation in Merino and Tukidale lines of sheep. Tukidale animals were chosen as a strong-woolled comparison to the fine-woolled Merino, due to their markedly different follicle densities and fibre characteristics. Different experimental procedures were utilised to provide a more thorough understanding of the role of the Notch signalling pathway in wool follicle initiation in sheep. DIG-labelled riboprobes for Delta-1, Delta-3 and Lunatic Fringe were generated from murine cDNA sequences and a PCR-generated ovine cDNA probe for Notch-1 provided information on gene expression using in situ hybridisation studies. Transcripts were detected in sectioned foetal skin prior to and during follicle development. In skin sampled prior to follicle initiation, Delta-l transcripts were detected throughout the epithelium and in some mesenchymal cells. Notch-l was detected in the epithelium, particularly in the basal layer, and in some mesenchymal cells. In skin sampled during follicle initiation, Delta-1 was expressed in the epithlium, basal layer of the epithelium and in some mesenchymal cells. Some Notch-l transcripts were detected in the epithelium, with staining appearing stronger in the basal layer. Notc-l transcripts were also detected in a few cells within the epithelial plug, in some mesenchymal cells and in the dermal condensates in the cells around the periphery of this structure. Counts of Delta-1 and Notch-1 labelled mesenchymal and dermal condensate cells were conducted. An interesting finding of this work was that no significant differences were detected between the numbers of cells labelled for Delta—1 or Notch-1 when the Merino and Tukidale skin samples were compared. Antibodies were used for immunohistochemistry studies in sectioned foetal sheep skin collected from Merino and Tukidale foetuses. Delta—1, Notch-1 and activated Notch protein expression were detected in skin samples. The behaviour of dermal papilla cells was investigated in vitro. An aggregating cell model whereby papilla cells were grown on the extracellular matrix complex MatrigelTM was developed. The cell cultures were shown to aggregate prior to reaching confluency, with the cells being observed over a 31 day period. A floating foetal sheep skin culture system was devised whereby skins were cultured on collagen-coated membranes for 4 days. Cell and skin cultures were examined in the presence and absence of the y-secretase inhibitor DAPT, which was dissolved in DMSO and added to the culture media. Dermal papilla cell aggregation was abolished in all dermal papilla cell cultures in the presence of 50uM DAPT. The formation of new follicle buds within skin harvested on day 70 of foetal development and cultured for 4 days was hindered. An investigation in to the effect of treatment with SOuM DAPT on follicle diameter in day 70 Merino skin cultures suggested that DMSO and DAPT had some effect on follicle diameter, when compared to the untreated controls. The results of this project have provided new insights into the mechanisms by which the wool follicle population is established in a foetus and suggest that some Notch signalling pathway genes are involved in the specification of prepapilla cell fate at follicle initiation.

Pancreatic neuroendocrine tumours (PNETs) are the most common neuroendocrine tumours diagnosed in humans and dogs. Due to the highly heterogeneous nature of these tumours, definitive data are still lacking over the molecular mechanisms involved in their cancerous behaviour. This study focused on insulinoma (INS), as it is the most commonly diagnosed PNET in human and veterinary oncology. INS is an insulin-producing tumour that causes a hypoglycaemic syndrome related to the excessive insulin production. In humans, it is often a small benign neoplasm readily curable by surgical resection whereas, in dogs, INS is often malignant. Despite current treatment modalities, malignant canine and human INS have a poor prognosis as patients tend to develop metastases in liver and lymph nodes that do not respond to current therapies. From a comparative oncology perspective, the close resemblance of canine and human malignant INS makes canine INS an interesting study model for human INS. Cancer stem cells (CSCs) are critical for the engraftment and chemoresistance of many tumours. Although CSCs have been isolated from a range of solid tumours, a comprehensive characterisation of INS CSCs has not yet been reported. In this study, it was confirmed that INS CSCs can be enriched and are potential targets for novel INS therapies. Highly invasive and tumourigenic human and canine INS CSCs were successfully isolated and exhibited greater resistance to chemotherapy, which may play a significant role in the poor prognosis of this disease. To date, the mechanisms by which tumours spread and the clinical causes of chemoresistance remain only partially understood. Here, RNA-sequencing analysis was performed over a small set of canine INS tumour samples in order to identify mechanisms involved in INS carcinogenesis through different stages of the disease. Preliminary data showed that distinct gene profiles characterised early and late stage of canine INS. Interestingly, differential gene expression and gene pathways analysis, highlighted that sets of genes involved in pancreatic embryogenesis and insulin secretion were overexpressed in canine primary INS lesions compared with normal pancreas. The Notch pathway is fundamental in pancreatic embryogenesis and it has been previously associated with carcinogenesis of neuroendocrine tumours and with the CSC phenotype. Protein analysis showed that the Notch pathway is activated in both human and canine INS CSCs, particularly when treated with chemotherapy, indicating that the Notch pathway may be involved in chemoresistance. Additionally, it was demonstrated that inhibition of the Notch pathway decreased INS CSCs' survival and chemoresistance, both in vitro and in vivo. These findings provide preclinical evidence that anti-Notch therapy may improve outcomes for patients with malignant INS.

The connections between cell cycle exit and terminal differentiation remain poorly understood. Cyclin dependent Kinase Inhibitors (CKIs) provide a possible link between entry into the quiescent state and differentiation. The initial aim of this project was to further investigate if the CKI p27Xic1 could promote differentiation in addition to, and independently of, its well characterised cell cycle exit function. p27Xic1 has been shown to be involved in cell fate determination during gliogenesis, neurogenesis, myogenesis and cardiogenesis and many mammalian Cip/ Kip CKI homologues of p27Xic1 have been described as important regulators of cellular processes beyond control of cell division. We aimed to investigate these roles during development of the embryonic kidney, the pronephros. We discovered that p27Xic1 does not affect differentiation during pronephrogenesis, but instead controls pronephric organ size through its cell cycle exit function. In addition we identified a previously unrecognised role for the cell cycle exit function of p27Xic1 in allocation of the somites during paraxial mesoderm segmentation. Preliminary results had suggested p27Xic1 expression in the pronephros was under the control of the Notch signalling pathway. Over-expressing a constitutively active form of Notch, Notch-ICD, and a dominant negative form of the Delta ligand, DeltaSTU, showed that both mis-activation and suppression of Notch signalling inhibited p27Xic1 expression. However, when investigating the effects these overexpressions had on pronephros development, we identified novel results indicating the Notch signalling pathway, which has previously been implicated in pronephros development, is essential for the separation of the proximal lateral and medial pronephric mesoderms. This process we propose is mediated by the Notch signalling pathway through the establishment of a boundary between these two distinct populations of cells, permitting both compartments to develop in isolation. The results in this thesis suggest novel mechanisms by which cell division controls X. laevis segmentation and organ size and how the Notch signalling pathway is able to pattern the pronephros anlagen such that the different compartments of the mature pronephros are able to develop, and thus function.

La conversion de la protéine prion cellulaire PrPC en une isoforme pathologique, la protéine prion scrapie PrPSc, est à l'origine d'un groupe de maladies neurodégénératives, les Encéphalopathies Spongiformes Transmissibles (EST). De nombreux travaux indiquent que la toxicité de la PrPSc implique une déviation de la fonction normale de la PrPC, cependant le rôle physiologique de la protéine prion n’est que partiellement compris. Dans ce travail, nous nous sommes attachés à identifier des voies de signalisation mobilisées par la PrPC qui pourraient à la fois rendre compte du rôle de cette protéine dans le développement du système nerveux et être impliquées dans la pathogénèse des EST. Nous montrons que la protéine prion contrôle l’activité de la voie Notch, une voie de signalisation qui joue un rôle majeur dans le développement mais également dans l’homéostasie du système nerveux central et la plasticité synaptique. Dans des modèles ex vivo et in vivo d’EST, nous mettons en évidence une diminution de l’activité de la voie Notch, ainsi que de l’expression des récepteurs de la famille Eph - connus pour leur implication dans l’activité synaptique. Cette diminution des Eph est retrouvée dans des cellules dépourvues de PrPC. Ainsi, l’observation d’un profil similaire entre la perte d’expression de la PrPC et l’infection par les prions renforce l’idée d’une déviation de la fonction normale de la PrPC par la PrPSc. Des inhibiteurs de l’activité histone désacétylase (HDAC) permettent de rétablir l’expression des acteurs de la voie Notch et des récepteurs Eph aussi bien dans les cellules déplétées en PrPC que dans celles infectées par les prions, suggérant que des mécanismes épigénétiques sont impliqués dans le contrôle transcriptionnel de ces gènes par la protéine prion. Ce travail fournit les bases pour évaluer un effet bénéfique des inhibiteurs de HDAC dans un modèle de souris infectées par les prions et ainsi déterminer si les HDAC pourraient constituer de nouvelles cibles thérapeutiques pour combattre les EST
The conversion of the cellular prion protein PrPC into a pathogenic isoform, the scrapie prion protein PrPSc, lies at the root of a group of neurodegenerative disorders known as Transmissible Spongiform Encephalopathies (TSEs). Several lines of evidence indicate that PrPSc-mediated toxicity involves a subversion of PrPC normal function, however, our knowledge of PrPC physiological role is still far from complete. In this work, we sought to identify signalling pathways mobilized by PrPC that could accommodate both its role in central nervous system development and its implication in TSE pathogenesis. We show that the prion protein controls the activity of the Notch pathway, which plays an overriding role during embryonic development as well as central nervous system homeostasis and synaptic plasticity. In both ex vivo and in vivo models of TSE, we monitored a decrease in Notch activity, together with reduced expression of Eph receptors, which are key players in synaptic activity. The reduction in Eph is also found in PrPC-depleted cells. Hence, our observation of a similar signature of PrPC depletion and prion infection strengthens the view that PrPSc diverts PrPC function. We found a restoration of Notch and Eph effectors expression in response to histone deacetylase (HDAC) inhibitors, both in PrPC-depleted and prion-infected cells, suggesting that epigenetic mechanisms are involved in the PrP-dependent transcriptional control of these genes. This work provides a foundation for assessing a beneficial effect of HDAC inhibition in prion-infected mice and thereby defining whether HDAC could represent novel therapeutic targets to combat TSEs

2012/2013
Cancer stem cells (CSCs) are proposed to be responsible for breast cancer heterogeneity, chemotherapeutic treatment failure, metastatic spread and disease recurrence. The precise identification of the molecular bases that govern the induction and maintenance of CSCs and their aggressive phenotypes is of utmost importance, since it may provide the rational to develop effective therapeutic strategies. In particular there is a considerable effort in finding common pathways, mutations or histological features that might be targeted for therapy, overcoming breast cancer heterogeneity. Here we now demonstrate that CSC self-renewal, chemoresistance, tumour growth and metastases formation capabilities’ are under direct control of Pin1’s enzymatic activity on the Notch signalling pathway. In particular Pin1 protects the nuclear activated forms of Notch1 and Notch4 (N1/4-ICD) from their E3-ubiquitin-ligase Fbxw7α, thereby boosting their protein levels and transcriptional activity. Fbxw7α acts as a potent inhibitor of CSCs maintenance by promoting protein degradation of N1- and N4-ICD, and, as a consequence, this ubiquitin-ligase strongly decreased tumour growth and metastases dissemination in vivo. Interestingly, concomitant over-expression of Pin1 almost completely recovered all these aggressive breast cancer traits. In tissues from breast cancer patients, we observed Notch signalling over-activation despite presence of the negative regulator Fbxw7α, which relied on high Pin1 protein levels. Notably, activation of the Notch-Pin1 axis correlated with poor prognosis in these patients. As a consequence of our findings, suppression of Pin1 holds promise in reverting aggressive phenotypes in breast cancer though shrinkage of CSCs number and a concomitant gain in chemosensitivity, carrying important implications for breast cancers therapy.
XXVI Ciclo
1985

The development of complex metazoans depends on the integration of a handful of signalling pathways that eventually modulate precise patterns of gene expression. The fact that just a few pathways are involved in the generation of such complexity in different organisms, suggests that these are highly regulated and conserved processes. The accurate spatio-temporal coordination of the signalling pathways controls the assignation of different cell fates and their patterning into tissues and organs. The source of diversity relies on the different possible interactions between signalling pathways, such as, the combination of signals and the order in which they are received by the cell or crosstalk. Due to their importance in development, abnormal signalling through these pathways has been strongly associated with developmental disorders, cancers and other diseases. The Notch and Wnt signalling pathways are key components of the intricate network that controls gene expression during development, and genetic analysis in Drosophila has highlighted that interactions between these two signalling pathways are important during this process.This thesis investigates the cross-regulatory interactions between Notch and Wnt signalling pathways in mammals. Using transcriptional reporter assays and biochemical analysis, I have found two molecular mechanisms underlying the inhibitory crosstalk between Notch and β-catenin, the effector of Wnt signalling pathway, in mammalian cells. At the membrane Notch inhibits β-catenin transcriptional activity through Deltex mediated endocytosis of Notch and a component required for β-catenin activation. This is similar to results observed in Drosophila. In the nucleus, I have identified a novel mechanism by which NICD-dependent transcription of Hes/Hey family of transcription factors prevents the activation of Wnt signalling pathway. This mechanism involves the formation of a physical complex between Hey1 and β-catenin/TCF, which allows Hey to block Wnt transcriptional activation. Additionally, I have found that these two mechanisms are conserved across vertebrates.Together the findings of this thesis improve our understanding of the molecular mechanism underlying the Notch/Wnt crosstalk. In turn, this will give an insight into unravelling how a handful of signalling pathways can generate sufficient diversity in signalling output to specify the hundreds of different cell fates generated to make a mammal. Elucidating these signalling networks will also contribute to our understanding of diseases, both their aetiology, by knowing how changes in one signal can influence another, and their treatment as mimicking points of crosstalk is likely to generate very specific therapeutic agents.

Worldwide, more than 1,000,000 women develop breast cancer each year, and more than 400,000 die because of it. Basal-like breast cancers, which account for 8% to 20% of all breast cancer cases, represent the most aggressive of breast cancers with the majority resistant to existing targeted therapies. Accumulating lines of evidence implicate the Notch pathway in the aetiology of these basal-like breast cancers; while current work in our lab supports the notion that signalling through the Ectodermal Dysplasin Receptor (EDAR) pathway is also important. Notch signalling functions in normal development to control cell fate decisions and is mediated primarily, although not exclusively, through the CBF1 / RBP-Jĸ transcription factor. Aberrant Notch signalling leads to mammary tumourigenesis in mice; however at the outset of this work it was unclear whether signalling through RBP-Jĸ and / or through alternative pathways is required. This thesis presents novel data showing that elevated Notch signalling through the RBP-Jĸ-dependent pathway alone in murine mammary glands causes a number of developmental defects, including reduced ductal outgrowth, increased ductal side branching at puberty and, most significantly, is sufficient to induce mammary tumourigenesis. The data presented also provide supporting evidence that Notch signalling through RBP-Jĸ likely contributes to tumourigenesis, at least in part, via the suppression of apoptosis. At the outset of this thesis far less was known regarding the role(s) of the EDAR pathway within the mammary gland. Despite its recognised function in the development of ectodermal appendages it has been predominantly studied in the context of hair and tooth development. We show here that elevated Edar signalling affects the morphology of numerous ectodermally-derived glands, including the mammary gland, where in general, it results in glands that are enlarged or more elaborately branched. Most significantly, we show that elevated Edar signalling causes mammary tumourigenesis in mice, and provide data to support the hypothesis that elevated EDAR signalling might also be important in a subset of basal-like breast cancers in humans. The murine mammary gland phenotypes seen in response to elevated Edar signalling, including the squamous metaplasia within Edar-induced tumours, are very similar to those observed when Wnt signalling is increased. We provide data to support a positive correlation between activation of the EDAR and Wnt pathways in murine mammary tumourigenesis and provide data to support a comparable interaction in the aetiology of basal-like breast cancer showing squamous differentiation in humans. In summary, this thesis identifies the EDAR pathway as a novel potential therapeutic target in the treatment of a subset of basal-like breast cancers, and provides evidence that signalling through the RBP-Jĸ-dependent Notch pathway is sufficient to induce mammary tumourigenesis, most likely through the suppression of apoptosis.

Notch3 signalling in vascular smooth muscle cell growth and survival VSMCs are the major components of the arterial wall. Under physiological conditions VSMCs exhibit a contractile phenotype, although possessing the capacity of phenotypic transition. The plasticity of VSMCs enables them to gain proliferative features which contribute to the pathologies of common vascular diseases in response to diverse stimuli. Hence, the stability of VSMCs in cell growth and survival is essential for maintaining proper arterial function. Diverse signalling pathways and growth factors have been identified as being important in the regulation of VSMC behaviour, and the Notch signalling is a relatively new participant. Proteins of Notch family are transmembrane receptors that transduce signals from neighbouring cells. Among the four Notch receptor subtypes, Notch3 is mainly expressed in VSMCs in small arteries. NOTCH3 gene mutation causes a human genetic stroke syndrome, CADASIL which is featured by systemic VSMC degeneration, suggesting the importance of Notch3 signalling in VSMC growth and survival. Although there is some knowledge for the Notch3 signalling in the regulation of VSMC proliferation and apoptosis, the molecular mechanisms underlying such regulation are largely unknown. By overexpressing the constitutive active form of human Notch3 (N3IC), it was confirmed that Notch3 promotes VSMC proliferation and protects VSMCs against apoptosis in primary rat aortic SMCs. It was also shown that Akt mediated both Notch3-induced VSMC proliferation and protection of serum-deprivation-induced apoptosis; while Erk 1/2 only contributed to Notch3-induced VSMC proliferation. In addition, the activation of PI3K/Akt was significantly blocked by PDGFR inhibitor, even in serum-free conditions, suggesting an intrinsic effect of Notch3 on PDGFR function without the requirement of the exogenous ligand, PDGF. It was also shown that Notch3 inhibits GSK-3α/ by phosphorylation and this effect was not blocked by the PI3K/Akt inhibitor, suggesting an alternative Akt-independent mechanism exists by which Notch3 regulates GSK-3 activity. Furthermore, using apoptosis pathway PCR arrays a number of apoptotic genes including BCL2 that were regulated by Notch3 activation in human VSMCs were identified. Notch3 also up-regulates the expression of its ligand Jagged1 in a PI3K/Akt dependent manner. Interestingly, Notch2 was significantly down-regulated and Notch1 was mildly upregulated by Notch3. In addition, an age-dependent increase in the expression of Notch3 in SMCs of the aortic root from 8 to 16 weeks old mice was identified, however, this declined in aged mice (24 weeks). These findings suggest a model for Notch3 function which is integrally involved in multiple signalling pathways in the regulation of VSMC growth and survival. It is hoped that these findings will contribute to the current knowledge of vascular biology and provide the basis for future studies on the development of potential therapeutic targets for cardiovascular diseases.

La myogenèse fœtale repose sur les cellules progénitrices musculaires PAX7+ qui assurent la croissance musculaire au cours du développement et qui sont à l’origine des cellules satellites. Nous avons cherché à interpréter les signaux régulant la myogenèse fœtale et leur lien avec le cycle cellulaire. Nous avons effectué une analyse exhaustive du cycle cellulaire des cellules myogéniques au cours de la myogenèse fœtale. Nous avons aussi identifié que les cellules PAX7+ progressant dans le cycle cellulaire (phases S, G2, et M) sont régionalisées aux extrémités des muscles. Les voies de signalisation BMP et NOTCH régulent positivement le nombre de cellules PAX7+ pendant le développement fœtal mais ont un effet différent sur la différenciation musculaire. Nous avons montré que les voies de signalisation BMP ou NOTCH augmentent le nombre de cellules PAX7+ de manière indépendante. Nous avons aussi identifié des interactions antagonistes entre ces deux voies lors de la différenciation musculaire. Nous avons testé l'importance de la contraction musculaire pendant la myogenèse fœtale chez l’embryon de poulet. Le blocage des contractions musculaires mime un phénotype de perte de fonction NOTCH, à savoir une diminution du nombre de cellules progénitrices musculaires avec une tendance à la différenciation musculaire. Nous avons aussi montré que les forces mécaniques produites par les contractions musculaires sont détectées par le co-activateur transcriptionnel YAP1 qui régule l'expression d’un ligand de NOTCH au sein des fibres musculaires, qui à son tour va maintenir le pool de cellules progénitrices musculaires fœtaux
Foetal myogenesis relies on PAX7+ muscle progenitors that provide the source of cells for muscle growth during development and for the generation of the satellite cell pool. We aimed to decipher the signals that regulate the balance between myogenic differentiation and proliferation. We performed an exhaustive analysis of the cell cycle phases of myogenic cells during foetal myogenesis. I defined that PAX7+ cells in the S/G2/M phases were enriched at the contact points to the tendons. BMP and NOTCH signals increase the number of PAX7+ cells during foetal development, but affect differentiation in a positive and negative manner, respectively. I revealed that BMP and NOTCH increase the number of PAX7+ cells independently of each other. However, they act antagonistically during differentiation. Thus, the interplay between NOTCH and BMP signalling differs in proliferation and differentiation. Because muscle is a mechanical tissue, we tested the importance of muscle contraction for foetal myogenesis in chick embryos. I found that the block of muscle contraction during foetal myogenesis mimicked a NOTCH loss-of-function, i.e. decreased the number of foetal muscle progenitors and shifted the balance between proliferation and differentiation towards a differentiation fate. Mechanical forces provided by muscle contractions are sensed in myonuclei by the transcriptional co-activator YAP1 that regulates expression of the NOTCH ligand JAGGED2 in muscle fibres. This JAGGED2 signal keeps the muscle progenitors in an undifferentiated state and suppresses differentiation

The Wnt and Notch signal transduction pathways are two important regulators of pituitary organogenesis. Wnt signalling regulates maintenance, proliferation and differentiation of the progenitor pool during early pituitary development, whilst Notch signalling prevents the premature specification of endocrine cell-types. Components of the Wnt and Notch pathways are downregulated in the pituitary as development proceeds. Furthermore, their expression is detected in the mature, adult gland. Our studies investigate the role of Wnt and Notch signalling in the adult and developing pituitary gland through genetic manipulation of signalling mediators in vivo. We investigate the function of Wnt signalling in committed POU1F1-expressing cells through the conditional deletion of the canonical Wnt signalling mediator Ctnnb1. We find that the loss of β-catenin from adult POU1F1-expressing cells does not dramatically affect adult pituitary hormone production, suggesting that the canonical Wnt pathway is not required for normal function of POU1F1- expressing cells. In parallel, we studied the role of Notch signalling in POU1F1-expressing cells through conditional overexpression of the constitutively activated Notch receptor. We find that, in contrast to previous studies, constitutive Notch signalling in POU1F1-expressing cells also does not affect their differentiation or function. At the same time, we do not find any pituitary-specific functions for the Notch signalling ligand Delta-like 1 homologue (DLK1). We therefore conclude that downregulation of Notch signalling is not required for normal development or function of POU1F1-expressing cells. Finally, our studies investigated the temporal specificity of Notch signalling throughout pituitary organogenesis through constitutive Notch activation in celltypes ranging from early progenitors to committed endocrine cells. Our results indicate that Notch signalling is especially important for specification of embryonic progenitors into melanotrophs. We also find evidence suggesting non-redundant functions for NOTCH receptor paralogues, and we ultimately propose a model for Notch signalling function during pituitary development.

Müller glia mediates retina regeneration in zebrafish. Despite exhibiting Müller glial with stem cell (hMSC) characteristics in vitro by the human retina, retinal regeneration mediated by Müller glia following disease or injury has not been demonstrated. Notch, Wnt, TGFβ and HBEGF signalling is well known to regulate retinal neurogenesis and inflammation, but the roles of these molecules in the neural differentiation of hMSC are not known. This thesis aimed to establish whether there is an interaction between these signalling pathways and the role that these factors play during retinal ganglion cell (RGC) and photoreceptor differentiation of hMSC. The research showed that inhibition of Notch signalling caused downregulation of components of the canonical Wnt signalling pathway in these cells, as demonstrated by a decrease in mRNA expression of the Wnt ligand WNT2B and its target genes WISP-1 and AXIN2. Addition of TGFβ1 did not significantly change the expression of the Notch signalling target HES1 or the RGC marker BRN3A/B. Culture of hMSC with a combination of factors that induce their photoreceptor differentiation (FGF2, taurine, retinoic acid and Insulin growth factor; collectively called FTRI), markedly upregulated the expression of components of the canonical Wnt signalling pathway, including WNT2B, DKK1 and active β-catenin. Although FTRI did not modify mRNA expression of WNT5B, a component of the non-canonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFβ1 not only decreased WNT2B expression, but inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, rhodopsin and recoverin. Inhibition of TGFβ1 signalling by an ALK5 inhibitor prevented TGFβ1 induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical Wnt signalling by XAV-939 prevented FTRI-induced photoreceptor differentiation. Similarly, HBEGF, a factor shown to be upregulated by FTRI also decreased Wnt signalling components such as WNT2B, WISP-1, DKK1 and AXIN2. Inhibition of HBEGF by its specific inhibitor CRM197 prevented photoreceptor differentiation. These observations suggest that both Notch and Wnt signalling pathways can regulate the neurogenicity of hMSC in vitro and that TGFβ as well as HBEGF play important roles in mediating key pathways leading to either RGC or photoreceptor differentiation of hMSC. Targeting components of both Notch and Wnt pathways may constitute targets for potential induction of endogenous regeneration of the human retina and this merits further studies.

The aim of this thesis was to examine role of Sirt1 in the Notch signalling pathway, using Drosophila as a model organism. Based on in vivo and in vitro studies, we conclude that Sirt1 plays a positive role in Notch signalling. In embryonic S2N cells, Sirt1 is responsible for the protection from metabolic stress-induced down-regulation of subset of E(Spl) genes. During development, Sirt1 is responsible for proper Notch-dependent specification of SOPs and wing development. Sirt1 can regulate the Notch signalling on multiple levels via deacetylation of various substrates involved in the Notch signalling revealed by our proteomic survey.

Seven genes involved in metabolism were tested as direct targets of the Notch signalling pathway. For each gene the occupancy of its enhancers by Su(H), its transcriptional response to Notch pathway and its biological functionality was verified in vitro and in vivo.

A dissertation submitted to the Faculty of Health Sciences, School of Clinical Medicine Department of Internal Medicine, University of The Witwatersrand, Johannesburg in fulfilment of the Degree of Masters of Science in Medicine Johannesburg 2015
The molecular pathology of triple negative breast cancer (TNBC) is poorly understood, consequently, no successful forms of therapy have been developed. Thus an improvement of knowledge and subsequently the discovery of novel treatments for the disease are imperative. It has been found that deregulation of the Notch signalling pathway promotes tumourigenesis in breast tissue. Therefore, it was of interest here to investigate whether the Notch signalling pathway is deregulated in TNBC and whether its abrogation affects the proliferation and migration of TNBC cell lines. The normal growth characteristics of MCF-7 cells (hormone sensitive) were compared to those of MDA-MB-231 and MDA-MB-436 (both hormone insensitive) cell lines and were determined by real-time cell impedance assays, using the ―xCELLigence‖ instrument. Thereafter, cells were treated with gamma secretase inhibitors (GSI) of the Notch signalling pathway. The MCF-7 cell line proliferated faster than the MDA-MB-231 and MDA-MB-436 cell lines. The proliferation of the MDA-MB-231 and MDA-MB-436 cell lines decreased significantly following treatment with inhibitors. Confocal microscopy was used to assess levels of the Notch intracellular component (a gamma secretase cleavage product) and E-cadherin (a breast tumour suppressor marker), pre- and post- treatment. Prior to drug treatments, confocal microscopy showed that the Notch intracellular component was highly expressed in the MDA-MB-231 cell line, and it was low in the MDA-MB-436 cell line, compared to the MCF-7 cell line. Following drug treatments, confocal microscopy showed a decreased expression of the Notch intracellular component in all three cell lines. Prior to drug treatment only the MCF-7 cell line expressed E-cadherin which was reduced post treatment. Subsequently the cell migration assays revealed that migration is reduced post- drug treatment in all three cell lines, despite no statistical significance. Overall the MDA-MB-231 and MDA-MB-436 cell lines were more significantly sensitive to the gamma secretase inhibitors compared to the MCF-7 cell lines. Therefore these observations suggest that the Notch signalling pathway is a plausible novel therapeutic target in the treatment of TNBC.

Developmental pathways such as Notch are known to regulate self-renewal and cell fate decisions in embryonic and adult tissue-specific stem cells, while deregulation of Notch signalling is associated with malignant transformation. Hypoxia develops in solid tumours due to rapidly dividing cancer cells, leaky blood vessels, and increasing distance from the nearest blood vessel. Tumour hypoxia is considered as malice, aiding cancer progression and hindering successful therapy by promoting EMT, stemness and drug resistance. Therefore, understanding signalling mechanisms regulated by hypoxia is therapeutically important. Notch pathway signalling is implicated in the maintenance of cancer stem/progenitor cells in a hypoxic tumour-microenvironment. Another protein activated in hypoxic condition is AMP-activated protein kinase (AMPK), a major player in energy homeostasis. Work done in the lab has implicated AMPK in regulating breast cancer stemness and drug resistance. Although independent studies have reported higher levels of activated AMPK and activated Notch1 in breast cancer, and hypoxia, which is prevalent in breast cancer, is shown to activate both pathways, a relationship between AMPK and Notch in breast cancer is yet to be established. These considerations led us to investigate whether AMPK facilitates cancer progression through the Notch pathway under hypoxic condition. The transmembrane Notch receptor is activated when exposed to membrane bound Jagged and Delta family ligands expressed on neighbouring cells. Ligand-receptor interaction results in two proteolytic cleavage events, producing Notch transmembrane (NTM) and release of Notch intracellular domain (NICD) from the plasma membrane. NICD is transported to the nucleus, where it forms a complex with the CSL DNA-binding protein and transcriptional co-activators. Nuclear NICD then activates expression of specific target genes. Our study revealed that activating AMPK through pharmacological agents or genetic approaches increased the accumulation of cleaved Notch1 protein (NTM and NICD) in invasive breast cancer cell lines such as MDA-MB-231, BT-474 and HCC-1806. An increase in cleaved Notch1 levels led to enhanced activation of Notch downstream target genes. Inhibiting AMPK activity or depleting AMPK reduced cleaved Notch1 levels in cell lines in-vitro and in BT-474 xenografts. Moreover, we show that AMPK positively modulates cleaved Notch1 protein levels by increasing its stability. Previous studies have demonstrated hypoxia-induced increase in cleaved Notch1 protein levels as well as stability, however, the mechanisms leading to this stabilization has remained unknown. We show that in hypoxia, activated AMPK is required for an increase in cleaved Notch1 stability. Mechanistically, we identified a reduction in interaction of Notch1 with its ubiquitin ligase Itch/AIP4, upon AMPK activation in hypoxia, thus, bringing about reduced ubiquitination and degradation of cleaved Notch1. Further, we identified AMPK-induced tyrosine phosphorylation of the ubiquitin ligase Itch/AIP4 under hypoxic conditions that disrupted Itch/AIP4-Notch1 interaction. The specific tyrosine kinase responsible was Fyn, a Src-family kinase member. Finally, we found that inhibition of AMPK in hypoxic cells affected Notch-mediated self-renewal adversely. In addition, we uncovered that inhibition of AMPK under hypoxia affected the hypoxia mediated increase in drug resistance. Together, these data suggested the involvement of hypoxia-AMPK-Notch1 axis in imparting self-renewal properties and drug resistance to breast cancer cells We have investigated the relevance of the signalling mechanism in patient derived breast cancer cells and interestingly, the hypoxia-AMPK-Itch/AIP4-Notch signalling axis is well conserved. More importantly, we assessed the concurrent activation of AMPK and Notch1 pathway in clinical breast cancer tissues which revealed that the activation of both these pathways correlated in large proportion breast cancer. Altogether, our study sheds light on the context-specific oncogenic role of AMPK, in reinforcing Notch1 signalling under hypoxia, to facilitate breast cancer progression. Based on our study, we propose that inhibition of AMPK can alleviate stem-like properties of breast cancer cells, thus rendering them susceptible to existing anti-cancer agents.

Tese de doutoramento, Biologia (Biologia Molecular), Universidade de Lisboa, Faculdade de Ciências, 2011
Bone marrow (BM)-derived cell populations participate in numerous physiological and pathological conditions in adult individuals, directly interacting with tissue specific cellular and extracellular matrix (ECM) components. In particular BM-derived endothelial progenitors (EPCs) contribute to physiological and pathological new vessel formation, a process termed posnatal-vasculogenesis. However the molecular mechanisms regulating their contribution are still lacking. In the present PhD thesis we adressed the role of two major pathways that regulate embryonic vasculogenesis: the Notch-Delta signalling pathway and Integrin-ECM pathway on EPC function during wound healing and tumor progression. To address the contribution of Integrin-ECM interactions to EPC function we tested the effect of the pro-angiogenic fibrin fragment E (FnE) on EPC vasculogenic properties. Our data shows that FnE potentiates the in vitro vasculogenic properties of EPCs, increasing their adhesion (via integrin α5β1), endothelial differentiation and paracrine factor production thus leading to increased in vivo wound vascularization and healing. Concerning the contribution of the Notch-Delta signalling pathway we defined that this pathway mediates bidirectional interactions between BM-derived cells and tissue resident cells during wound healing and tumor progression. In detail we observed that activation of the Notch pathway on EPCs increases integrin α3β1 expression, improving EPC adhesion and endothelial differentiation as well as their pro-angiogenic and wound healing potential in vitro and in vivo. In a tumor setting we observed that EPC-mediated activation of the Notch signaling on ECs regulates vessel stability by increasing the expression of basement membrane components as well as endothelial cell-cell junction proteins, thus being essential for neo-vessel normalization during tumor growth. Further characterization of BMderived cells recruited to tumor tissues lead us to the identification of BM-derived tumor associated macrophages (TAMs) that can activate Notch signaling on colon carcinoma cells inducing tumor epithelial to mesenchymal transition (EMT) thus contributing to tumor progression and metastisation. Taken together our results suggests that both Integrin-ECM and Notch-Delta pathways modulate the properties of BM-derived populations, being essential for the vasculogenic properties of EPCs and for the tumor promoting functions of TAMs, thus making these pathways valid targets in both wound healing and tumor progression.
O sistema cardiovascular constitui o primeiro sistema de órgãos que se forma durante o desenvolvimento embrionário dos vertebrados, tendo como principais funções oxigenação e nutrição de todos os tecidos assim como a remoção de produtos secundários do metabolismo e regulação térmica via vasoconstrição e dilatação. Os principais componentes do sistema cardiovascular são o coração, o sangue e os vasos sanguíneos. Por sua vez os vasos sanguíneos têm como unidade funcional e estrutural a célula endotelial (CE) ao nível da qual são feitas as trocas celulares, metabólicas e gasosas entre as células de todos os tecidos e o sangue. A formação de vasos sanguíneos ocorre durante o desenvolvimento embrionário sendo um processo extremamente complexo que envolve essencialmente dois mecanismos celulares distintos mas interligados: vasculogénese e angiogénese. A vasculogénese é o processo através do qual células precursoras endoteliais, denominadas angioblastos, migram, agregam-se e coalescem formando uma rede primitiva de tubos endoteliais. O subsequente crescimento, expansão e estabilização dessa rede vascular primitiva ocorre através da proliferação, migração e consequente ramificação de CE activadas, num processo denominado angiogénese. Num organismo adulto as CE, assim como os vasos sanguíneos que elas constituem encontramse num estado não proliferativo, denominado quiescência. No entanto, na presença de estímulos específicos nomeadamente em situações fisiológicas (cicatrização de feridas, revascularização de tecidos isquemicos, ovulação, menstruação ou gravidez) ou patológicas (cancro, psoriase, artrite, retinopatias, aterosclerose ou em feridas com cicatrização atrasada) as CE podem tornar-se activas e por angiogénese geram novos vasos sanguíneos. No entanto dados recolhidos na ultima década sugerem que à semelhança do que acontece durante o desenvolvimento embrionário, também no adulto novos vasos sanguíneos se podem formar por vasculogénese. Efectivamente foram identificados em indivíduos adultos células denominadas progenitores endoteliais (PE), que partilham semelhanças com angioblastos embrionários, e que em situações fisiológicas e patológicas são recrutados e diferenciam-se em CE, que por sua vez incorporam os novos vasos formados, num processo denominado vasculogénese pós-natal. PE são definidos como células com origem na medula óssea e que são caracterizados pela expressão de marcadores moleculares específicos de células estaminais como o CD133, CD34 ou o Sca-1 (em ratinho) assim como marcadores endoteliais como o VEGFR-2 (receptor 2 do factor de crescimento vascular). Em resposta a estímulos específicos provenientes de tecidos em remodelação vascular os PE são mobilizados da medula óssea para a circulação, onde são transportados até atingirem os locais de remodelação vascular onde abandonam a circulação por extravasão, invadem os tecidos adjacentes e eventualmente diferenciam-se em CE incorporando os novos vasos formados. O processo de diferenciação endotelial dos PE pode ser dividido em 3 fases essenciais: adesão, mediada por integrinas, dos PE aos componentes da matriz extracelular, sobrevivência e proliferação dos PE aderentes em resposta a factores de crescimento e a sinais da matriz extracelular e finalmente aquisição de marcadores e propriedades endoteliais. Apesar de já ser conhecida e descrita a contribuição dos PE em diversas condições fisiológicas e patológicas os mecanismos que regulam a sua diferenciação em CE ainda são desconhecidos. Assim sendo os objectivos desta tese de doutoramento são a compreensão dos mecanismos moleculares envolvidos na diferenciação endotelial, em contexto de cicatrização de feridas e de progressão tumoral, com especial foco em vias que são essenciais para a vasculogénese embrionária: via de interacção entre integrinas e proteínas da matriz extracelular e via de sinalização Notch-Delta. Além da compreensão dos mecanismos moleculares que regulam a diferenciação endotelial em PE também foi feita investigação relativa ao papel da via de sinalização Notch-Delta na comunicação entre outras populações celulares derivadas da medula óssea (além dos PE) e células tumorais, durante a progressão tumoral. No capítulo 3 desta tese de doutoramento abordámos o papel da matriz extracelular provisória composta essencialmente por fibrina e pelos seus fragmentos de degradação, que se forma durante a cicatrização de feridas, na função dos PE. Em particular caracterizámos in vitro o papel do fragmento de degradação da fibrina E (FnE), que tem potentes propriedades pró-angiogénicas, na diferenciação e nas propriedades vasculogénicas dos PE. De acordo com os nossos resultados FnE potencia a adesão dos PE, via integrina α5β1, assim como a sua diferenciação endotelial comparativamente a outros componentes da matriz extracelular. Surpreendentemente observámos que PE cultivados em FnE produzem níveis mais elevados de factores paracrinos, nomeadamente de factores pró-angiogénicos e de inúmeras citoquinas. Utilização de meios condicionados de PE cultivados em diferentes componentes da matriz revelou que os meios condicionados dos PE na presença de FnE têm propriedades pró angiogénicas assim como propriedades quimiatractoras de monócitos sanguíneos extremamente elevados comparativamente às restantes condições. Para validarmos o papel pró-vasculogénico do FnE em PE in vivo, utilizámos uma matriz sintética enriquecida com FnE (denominada Smart Matrix – SM) em modelos murinos de cicatrização de feridas. De acordo com os nossos resultados a adição de SM conjuntamente com PE potenciou significativamente o fecho das feridas, estando associado a uma maior vascularização da ferida comparativamente com as restantes condições. Em conclusão os resultados apresentados no capítulo 3 sugerem que FnE tem grandes propriedades vasculogénicas, pelo que a sua administração sob a forma de uma matriz sintética conjuntamente com PE representa uma possível abordagem terapêutica para potenciar a cicatrização de feridas quer resultantes de trauma, queimadura ou ulceração crónica resultante de diabetes, pressão ou estase venosa. No capítulo 4 investigámos o papel da via de sinalização Notch-Delta na diferenciação endotelial e nas propriedades vasculogénicas dos PE, num contexto de cicatrização de feridas. De acordo com os nossos resultados os PE expressam ligandos e receptores da via Notch-Delta e durante a diferenciação endotelial destas células observámos um aumento na activação da via Notch-Delta. Curiosamente a inibição farmacológica da via Notch-Delta levou a uma redução drástica na adesão dos PE a diversos componentes das matriz extracelular assim como a uma redução significativa do número de CE obtidas no final do ensaio de diferenciação endotelial in vitro. Mecanisticamente observámos que inibição da via Notch-Delta nos PE conduzia a uma redução da expressão da integrina α3β1, razão pela qual observámos redução na adesão e consequentemente na diferenciação dos PE. Adicionalmente verificámos que inibição farmacológica da via Notch-Delta nos PE reduzia a capacidade destas células induzirem angiogénese e migração endotelial em ensaios in vitro, sugerindo que a via Notch-Delta é essencial não so para a adesão e diferenciação endotelial dos PE mas também para as suas propriedades pró-angiogenicas. Após constatarmos o papel essencial da via Notch-Delta nos PE, testámos o efeito da inibição desta via em PE em modelos murinos de cicatrização de feridas. Em confirmação dos resultados in vitro observámos que ratinhos injectados com PE normais têm um aumento da taxa de cicatrização associado a um aumento do numero de vasos sanguíneos presentes na ferida. Por outro lado o efeito dos PE na cicatrização e vascularização perde-se quando a via Notch-Delta é inibida, curiosamente PE com a via Notch-Delta inibida estão presentes na ferida em frequências menores que os PE normais. Estes dados demonstram que a activação da via Notch-Delta em PE potencia as suas propriedades vasculogénicas e próangogénicas, sendo essencial para a sua capacidade de acelerar a cicatrização de feridas em modelos murinos. No capitulo 5 abordámos o papel de um dos ligandos da via Notch-Delta expresso em PE, o Dll4 (Delta-like ligand 4) na angiogénese tumoral. De acordo com os nossos resultados a expressão de Dll4 em PE é regulada directamente por 2 factores mobilizadores de PE: VEGF (factor de crescimento vascular) e SDF-1 (factor derivado do estroma de medula óssea). Para testar o efeito de Dll4 na activação da via Notch-Delta em CE, co-cultivámo PE com diferentes níveis de expressão de Dll4 conjuntamente com CE e posteriormente analisámos os genes expressos diferencialmente nas CE consoante os níveis de Dll4 dos PE. De acordo com a nossa análise a activação da via Notch-Delta nas CE via Dll4 expresso pelos PE conduziu ao aumento da expressão de componentes da matriz extracelular (nomeadamente fibronectina) e de componentes das adesões endoteliais (ICAM2 e VE-Caderina). In vivo observámos que em ratinhos transplantados com PE heterozigóticos para Dll4 (Dll4+/-) o crescimento tumoral era reduzido relativamente a ratinhos transplantados com PE normais, no entanto analise do tecido tumoral revelou que os tumores dos ratinhos transplantados com PE Dll4+/- tinham maior densidade vascular, mas menor proliferação, maior frequência de células apoptóticas e áreas de hipoxia mais extensas relativamente aos tumores crescidos em ratinhos transplantados com PE normais. Esta observação sugeriu que os vasos tumorais desenvolvidos em ratinhos transplantados com PE Dll4+/- eram mais instáveis embora existissem em maior numero. De facto análise histológica revelou que estes vasos apresentam menor expressão de fibronectina e menor cobertura com pericitos (células vasculares musculares), confirmando que a activação da via Notch-Delta em CE via Dll4 expresso por PE é essencial para a estabilização da vasculatura tumoral nascente e consequentemente para o crescimento tumoral. Finalmente para validar o efeito de Dll4 expresso nos PE na estabilização da vasculatura tumoral transplantámos ratinhos com PE com sobre-expressão de Dll4 e analisámos a vasculatura tumoral. De facto observámos que embora o crescimento tumoral não tenha sido alterado, assim como o numero total de vasos formados, a quantidade de fibronectina assim como o calibre dos vasos tumorais era superior nos ratinhos transplantados com PE com sobre-expressão de Dll4 do que nos ratinhos transplantados com PE normais, confirmando que a activação da via Notch-Delta em CE via Dll4 expresso em PE conduz à estabilização vascular. Concluindo, os dados apresentados no capitulo 5 descrevem o papel essencial de Dll4 expresso por PE na activação da via Notch-Delta em CE, conduzindo ao aumento de proteínas da matriz extracelular assim como de componentes das junções celulares entre células endoteliais essenciais para a estabilização e funcionalidade da vasculatura tumoral. Finalmente no capítulo 6 investigámos a contribuição de outras populações celulares derivadas da medula óssea para a progressão tumoral. Para este efeito utilizámos ratinhos nos quais transplantámos medula óssea proveniente de ratinhos actina-GFP, pelo que obtivemos ratinhos nos quais todas as células na medula expressavam GFP. Posteriormente inoculámos estes ratinhos com tumores derivados de linhas celulares isoladas de carcinoma de cólon (HCT15) e procedemos à analise histológica dos tumores, após estes crescerem durante 1 ou 4 semanas (tumores precoces e tardios). Análise histológica revelou que em tumores tardios há uma contribuição significativa de células derivadas da medula óssea (GFP+) e que estas células expressam essencialmente marcadores mielóides (CD11b). Inesperadamente constatámos que nas regiões tumorais onde as células derivadas da medula óssea se acumulavam, as células tumorais perdiam a expressão de marcadores epiteliais (nomeadamente a E-Caderina), adquirindo a expressão de marcadores mesenquimatosos (nomeadamente Vimentina) sugerindo que estas células tumorais estavam em transição epitelial-mesenquimal (TEM). Para confirmar a suspeita que células derivadas da medula óssea poderiam estar a induzir TEM em células tumorais de carcinoma do cólon, procedemos a co-culturas in vitro com células derivadas da medula associadas ao tumor com células do carcinoma do cólon. Surpreendentemente verificámos que células derivadas da medula óssea associadas ao tumor tinham a capacidade de induzir TEM em células tumorais e ainda que essa capacidade dependia da activação da via Notch-Delta nas células tumorais. Após uma caracterização detalhada da população de células derivadas da medula associadas ao tumor capazes de induzir TEM nas células tumorais, definimos esta população como sendo constituída por macrófagos associados a tumor ou MAT (CD11b+ F4/80+) que expressava o ligando da via Notch Jagged-2. Experiências de co-cultura in vitro desta população com células tumorais confirmaram que esta população consegue via Jagged-2 induzir a activação da via Notch-Delta promovendo TEM nas células tumorais sendo essencial para a progressão tumoral. Em conjunto os dados apresentados no capitulo 6 sugerem que durante a progressão tumoral, populações de células derivadas da medula podem de facto modular propriedades das células tumorais induzindo TEM e consequentemente induzindo um fenotipo mais invasivo e potencialmente mais metastático nos tumores primários. Em conclusão, o trabalho realizado neste projecto de doutoramento permitiu atribuir à via Notch- Delta e também as interacções entre integrinas e proteínas da matrix extracelular um papel essencial na comunicação entre populações celulares derivadas da medula óssea, em particular PE e MAT, e componentes celulares e da matrix extracelular presentes durante a cicatrização de feridas ou durante a progressão tumoral, sugerindo assim o uso destas vias e destas populações como potenciais alvos terapêuticos.
Fundação para a Ciência e a Tecnologia (FCT, SFRH / BD / 31381 / 2006)

Tese de mestrado, Ciências Biomédicas, Universidade de Lisboa, Faculdade de Farmácia, 2016
Previous studies have accomplished direct lineage reprogramming of many cell types to different ones by using defined combinations of transcription factors. Vierbuchen et al. showed that the combined ectopic expression of Ascl1, Brn2 and MyT1L can efficiently reprogram mouse embryonic fibroblasts (MEFs) into induced neuronal (iN) cells. In another study, Ascl1 was characterized as the main driver of this process by its activity as a pioneer factor. Previous experiments with Ascl1 single-reprogramming showed that Ascl1 is capable of converting MEFs into iN cells, although the reprogrammed neurons show low levels of maturity. On the other hand, several reprogramming experiments associated MyT1L with a late function by promoting the maturation of iN cells, but not with the capacity to reprogram MEFs into iN cells like Ascl1. However, a mechanistic characterization of MyT1L still needed to be clarified. MyT1L is a member of the MYT1 family, also including MyT1 and MyT3, all zinc-finger transcription factors. Recent work from our laboratory showed that MyT1, a paralog of MyT1L, acts as a repressor of Notch targets, in neural stem/progenitor cells. One of those identified Notch targets was Hes1. In neurogenesis, the Notch pathway induces the activation of the Notch downstream effector Hes1. Hes1 functions as a repressor of proneural genes, such as Ascl1, as well as their target genes. Similar to the neurogenesis context, it is tempting to speculate that in Ascl1-dependent reprogramming Hes1 may be functioning as a repressor of Ascl1 targets in MEFs. The goal of this work is to investigate the role of MyT1L and the Notch signalling pathway in Ascl1-dependent reprogramming of MEFs into iN cells. To evaluate Notch activity in MEFs, I compared the expression levels of two Notch targets, Hes1 and Hes5, between MEFs and neural stem cells. I show that Hes1 expression in MEFs is similar to Hes1 expression in neural stem cells. Hes5 expression is substantially lower in MEFs than in neural stem cells. This suggests low Notch activity in MEFs as previous studies identify the Hes5 promoter as readout of Notch activation. Chemical inhibition of Notch signalling did not alter the Hes1 expression in MEFs. I show that the proximal promoter region of Hes1, that mediates regulation by Notch and MyT1 in neural stem/progenitor cells, is accessible to transcription factor binding in MEFs. Additionally, I show that the Notch effector transcription factor RBPJ binds to the Hes1 proximal promoter region. These results in conjunction with the high levels of Hes1 expression in MEFs suggest that the Notch pathway is not the main regulator of Hes1 expression in these cells. Work from our laboratory showed that, in transcriptional assays, MyT1 represses the Hes1 proximal promoter activity, after Notch activation. Here I show that Myt1L can counteract the Notch activation of the Hes1 promoter in a transcriptional assay. The Hes1 proximal promoter contains three consensus binding sites of the MYT1 family suggesting that MyT1L regulates the Hes1 promoter by direct DNA-binding to this region. Using chromatin immunoprecipitation assay against a tagged version of Myt1L, I show that MyT1L directly binds to the Hes1 promoter region two days after being ectopically expressed in MEFs. Finally I started the optimization of the Ascl1-depedent reprogramming protocol in MEFs. I did observe reprogrammed iN cells after single or combined expression of Ascl1 or Ascl1/MyT1L, respectively. However, the percentage of iN cells to total number of cells in culture revealed low reprogramming efficiency. Additionally, iN cells observed show low levels of maturity in single or combined expression of Ascl1 or Ascl1/MyT1L. Nonetheless, this protocol still needs further improvement. Overall, my findings indicate that MyT1L binds to DNA in MEFs at early stages of the Ascl1-dependent reprogramming protocol. The results suggest that MyT1L represses the expression of Hes1 in Ascl1-dependent reprogramming and this may lead to the activation of the Ascl1 targets that promote iN cell maturation.
Vários estudos têm vindo a demonstrar que a reprogramação directa de uma linha celular somática para outros tipos celulares pode ser alcançada através da expressão ectópica de factores de transcrição. De facto, trabalho desenvolvido por Yamanaka e Takahashi (Takahashi and Yamanaka, 2006) demonstrou que a adição de quatro factores de transcrição é suficiente para reprogramar fibroblastos em células estaminais pluripotentes. Este estudo estabeleceu uma mudança de paradigma na forma como olhamos para o programa de transcrição e a plasticidade do genoma da célula. A reprogramação de um tipo celular a partir de células estaminais ou somáticas oferece um enorme potencial de aplicações na medicina regenerativa e na terapia de doenças. A reprogramação de fibroblastos em células neuronais foi alcançada através da adição de três factores de transcrição, Brn2, Ascl1 e MyT1L (BAM) (Vierbuchen et al., 2010), em que o Ascl1 é o factor de transcrição principal, uma vez que, sozinho, é capaz de converter os fibroblastos em neurónios, apesar de apresentarem baixa complexidade morfológica e capacidade funcional (Chanda et al., 2014). Curiosamente, Ascl1 funciona como um factor pioneiro, sendo capaz de se associar às regiões genómicas, independentemente de se encontrarem em locais de cromatina acessível (Raposo et al., 2015; Wapinski et al., 2013). O Ascl1 é um factor de transcrição proneural que actua como um regulador da diferenciação neuronal no cérebro de mamíferos (Bertrand et al., 2002; Wilkinson et al., 2013). No processo de neurogénese, Ascl1 actua principalmente como um activador de transcrição sobre uma grande variedade de genes que controlam vários passos da neurogénese, como a proliferação das células estaminais neurais/progenitoras, migração celular e crescimento das neurites (Borromeo et al., 2014; Castro et al., 2011, 2006). Recentemente, Ascl1 foi identificado como um factor de transcrição capaz de modificar a cromatina dos seus genes alvos, durante a neurogénese, promovendo a acessibilidade da cromatina para Ascl1 (Raposo et al., 2015). Durante a neurogénese, o Ascl1 é regulado pela via de sinalização Notch. No desenvolvimento do sistema nervoso, a via de sinalização Notch é responsável pela manutenção da população de células estaminais neuronais/progenitoras, através da inibição da diferenciação neuronal. A proteína Notch activa a expressão de genes repressores da neurogénese, dos quais se incluem os genes Hes1 e Hes5. Os genes Hes1/5 actuam como repressores da transcrição, sendo um dos seus alvos Asc1. Adicionalmente, resultados anteriores do nosso laboratório demonstraram que Hes1 inibe a expressão dos genes alvos de Ascl1. Recentemente, estudos realizados no nosso laboratório revelaram que um alvo de Ascl1 durante a neurogénese, o factor MyT1, tem um papel importante em bloquear a expressão de genes alvo de Notch, em particular o Hes1. MyT1 é um factor de transcrição da família MYT1, que é composta por outros 2 factores de transcrição: MyT1L e MyT3. Os membros desta família são altamente homólogos, particularmente nos domínios proteicos zinc-fingers, responsáveis pela ligação ao ADN (Bellefroid et al., 1996; Kim et al., 1997). Todos os membros da família MYT1 são expressos no desenvolvimento do sistema nervoso central. Em particular, MyT1L é expresso exclusivamente em neurónios e é detectado tanto na neurogénese como na fase adulta do organismo (Matsushita et al., 2014). Na reprogramação neuronal, o MyT1L tem sido utilizado em vários protocolos para promover um aumento da complexidade morfológica e das propriedades electrofisiológicas das células neuronais (Ambasudhan et al., 2011; Pang et al., 2011; Vierbuchen et al., 2010; Yoo et al., 2011). Considerando os resultados do nosso laboratório em que se demonstrou que MyT1 é um repressor da expressão de Hes1, colocámos a hipótese de que MyT1L pudesse também actuar na reprogramação de células neuronais como um repressor da expressão de Hes1. O trabalho desta dissertação teve como objectivo investigar o papel do MyT1L e da via de sinalização Notch na reprogramação de fibroblastos em células neuronais promovida por Ascl1. Em primeiro lugar analisei a actividade da via de sinalização Notch nos fibroblastos através da análise de expressão de dois genes alvos de Notch, Hes1 e Hes5. A expressão destes genes foi comparada entre fibroblastos e células NS-5, uma linha de células estaminais neurais com elevada actividade da via Notch. Os resultados demonstraram que o nível de expressão de Hes1 em fibroblastos e em células NS-5 são semelhantes. No entanto, após inibição química da actividade de Notch não observei nenhuma alteração na expressão de Hes1, o que sugere que a via sinalização Notch não é a principal reguladora de Hes1 nos fibroblastos. Contrariamente a Hes1, a expressão de Hes5 é consideravelmente inferior nos fibroblastos em relação às células NS-5. Por outro lado, a inibição química da actividade de Notch levou a uma diminuição da actividade da expressão de Hes5, indicando que Hes5 é regulado por Notch em fibroblastos. Em segundo lugar, investiguei qual o possível papel de MyT1L na regulação da expressão de Hes1 em fibroblastos. Analisei que a região promotora de Hes1, onde anteriormente o nosso laboratório demonstrou haver associação de MyT1 em células neurais/progenitoras estaminais, se encontra com cromatina acessível à associação de factores de trancrição, em fibroblastos. Também analisei a actividade de MyT1L nessa região promotora de Hes1 através de um ensaio de transcrição com a co-expressão de MyT1L e receptor Notch1 activado. Esta análise revelou que o MyT1L é um repressor do promotor de Hes1, dependente da activação pela via Notch. Esta região promotora de Hes1 contém três sítios de ligação ao ADN comum à família MYT1. De facto, os resultados da imunoprecipitação da cromatina extraída de fibroblastos revelaram uma associação do MyT1L ectopicamente expresso na região promotora de Hes1. Hes1 é um factor repressor da expressão de Ascl1 e dos seus genes alvos. De facto, é possível que, no contexto da reprogramação promovida por Ascl1, os níveis de Hes1 endógeno possam estar a reprimir a expressão dos genes alvos de Ascl1. Esta repressão de Hes1 pode explicar o baixo nível de diferenciação das células neuronais observado na reprogramação com apenas sobre-expressão de Ascl1. De facto, MyT1L foi descrito como tendo um papel importante no desenvolvimento de características de neurónios morfologicamente complexos. Assim é possível que, o MyT1L promova indirectamente a expressão dos genes alvos de Ascl1, através da inibição da expressão de Hes1. Deste modo, o protocolo de reprogramação de fibroblastos em células neuronais mediado por Ascl1 foi optimizado, com o objectivo de investigar a interacção de MyT1L e Hes1, no contexto desta reprogramação. Infelizmente, o protocolo não foi estabelecido com sucesso, devido, a uma elevada taxa de morte célular. Apesar da elevada morte celular, consegui obter células neuronais, a partir de fibroblastos, com apenas a sobre-expressão de Ascl1 em co-expressão com MyT1L. As células neuronais obtidas com estas duas condições apresentavam baixos níveis de complexidade morfológica. Em conclusão, demonstrei que MyT1L encontra-se associado à região promotora de Hes1 quando expresso de modo ectópico em fibroblastos e que Myt1L actua como um repressor da actividade da região promotora de Hes1 promovida pela activação da via de sinalização Notch. A junção destes dois resultados sugere que a inibição da expressão de Hes1 se reflicta nos fibroblastos após sobre-expressão de Myt1L. A função de MyT1L pode incluir a repressão da expressão de Hes1, promovendo a activação de genes alvos de Ascl1 responsáveis pela maturação neuronal. Experiências futuras que demonstrem uma diminuição da expressão de Hes1 após a sobre-expressão de MyT1L em fibroblastos devem ser consideradas. Adicionalmente, futuras experiências devem também focar-se na descoberta de outros genes alvo de MyT1L em fibroblastos que possam ter um papel importante na reprogramação promovida por Ascl1 de fibroblastos em neurónios.
The studies presented in this thesis were carried out at the Instituto Gulbenkian Ciência (IGC) at the Molecular Neurobiology Laboratory, Oeiras. The present study was supported by Fundação Calouste Gulbenkian.

Lors d’une infection par un pathogène, des lymphocytes T CD8+ naïfs (LTn) spécifiques de l’antigène sont activés, prolifèrent et se différencient en LT effecteurs (LTe). Les LTe produisent différentes cytokines et acquièrent une activité cytotoxique menant à l’élimination du pathogène. Seulement 5 à 10 % des LTe survivront et se différencieront en LT mémoires (LTm), qui sont capables de répondre plus rapidement lors d’une seconde infection par le même pathogène, contribuant au succès de la vaccination. Toutefois, la compréhension de l’ensemble des mécanismes régulant le développement des LTe et des LTm demeure incomplète. Afin de mieux comprendre les signaux requis pour la différenciation des LT CD8+ lors de la réponse immune, nous avons posé deux hypothèses. Nous avons d’abord proposé que différentes cellules présentatrices d’antigène (CPA) fournissent différents signaux au moment de la reconnaissance antigénique influençant ainsi le devenir des LT CD8+. Vu leur potentiel d’utilisation en immunothérapie, nous avons comparé la capacité d’activation des LT CD8+ par les lymphocytes B activés via le CD40 (CD40-B) et les cellules dendritiques (CD). Nous avons montré que l’immunisation avec des CD40-B induit une réponse effectrice mais, contrairement à l’immunisation avec des CD, pratiquement aucun LTm n’est généré. Les LTe générés sont fonctionnels puisqu’ils sécrètent des cytokines, ont une activité cytotoxique et contrôlent une infection avec Listeria monocytogenes (Lm). Nous proposons qu’une sécrétion plus faible de cytokines par les CD40 B ainsi qu’une interaction plus courte et moins intime avec les LT CD8+ comparativement aux CD contribuent au défaut de différenciation des LTm observé lors de la vaccination avec les CD40-B. Ensuite, nous posé l’hypothèse que, parmi les signaux fournis par les CPA au moment de la reconnaissance antigénique, la voie de signalisation Notch influence le développement des LTe, mais aussi des LTm CD8+ en instaurant un programme génétique particulier. D’abord, grâce à un système in vitro, le rôle de la signalisation Notch dans les moments précoces suivant l’activation du LT CD8+ a été étudié. Ce système nous a permis de démontrer que la voie de signalisation Notch régule directement l’expression de la molécule PD-1. Ensuite, grâce à des souris où il y a délétion des récepteurs Notch1 et Notch2 seulement chez les LT CD8+ matures, un rôle de la voie de signalisation Notch dans la réponse immune des LT CD8+ a été démontré. Nos résultats démontrent que suite à une infection avec Lm ou à une immunisation avec des CD, la signalisation Notch favorise le développement de LTe, exprimant fortement KLRG1 et faiblement CD127, destinés à mourir par apoptose. Toutefois, la signalisation Notch n’a pas influencé la génération de LTm. De façon très intéressante, l’expression des récepteurs Notch influence la production d’IFN- en fonction du contexte d’activation. En effet, suite à une infection avec Lm, l’absence des récepteurs Notch n’affecte pas la production d’IFN- par les LTe, alors qu’elle est diminuée suite à une immunisation avec des CD suggérant un rôle dépendant du contexte pour la voie de signalisation Notch. Nos résultats permettent une meilleure compréhension des signaux fournis par les différentes CPA et de la voie de signalisation Notch, donc des mécanismes moléculaires régulant la différenciation des LT CD8+ lors de la réponse immunitaire, ce qui pourrait ultimement permettre d’améliorer les stratégies de vaccination.
Following an infection with a pathogen, antigen-specific naive CD8+ T lymphocytes (Tn) will proliferate and differentiate into effector (Te) cells. Those Te cells will produce different cytokines and acquire a cytotoxic activity, leading to pathogen clearance. Only 5 to 10 % of Te cells will survive and differentiate into memory CD8+ T lymphocytes (Tm) able to respond rapidly following a second encounter with the same pathogen, contributing to the success of vaccination. However, the mechanisms regulating Te and Tm cells development remain incompletely understood. To better understand the signals required for CD8+ T lymphocytes during an immune response, we proposed two hypotheses. First, we propose that different antigen presenting cells (APCs) can deliver different signals to CD8+ T lymphocytes at the time of priming leading to different outcome. Given their potential for use in immunotherapy, we compared the ability of CD40 activated B lymphocytes (CD40-B) and dendritic cells (DCs) to activate CD8+ T lymphocytes. We have shown that CD40-B cell immunisation leads to an effector response but very few Tm cells are generated compared to DC immunisation. The Te cells generated following CD40-B cell immunisation are functional because they secrete cytokine, are cytotoxic and control a Listeria monocytogenes (Lm) infection. We propose that CD40-B cells secrete less cytokines and interact during shorter period of time with the CD8+ T lymphocytes, without engulfment, contributing to the decreased Tm generation observed following immunisation with CD40-B cells. Second, among the signals provided by APC at the time of CD8+ T lymphocyte priming, we have hypothesised that the Notch signalling pathway influences Te and Tm cell differentiation by inducing a particular genetic program. Using an in vitro system, we first studied the role of the Notch signalling pathway in the hours following CD8+ T lymphocyte priming. We demonstrated that Notch signalling directly regulates PD-1 expression. Then, studying mice where Notch1 and Notch2 receptor genes are deleted only in mature CD8+ T lymphocytes, we characterised the role of the Notch signalling pathway on Te and Tm differentiation during an immune response. Our results show that following Lm infection or a DC immunisation, the Notch signalling pathway promotes the differentiation of short lived effector cells Te cells (KLRG1highCD127low) meant to die by apoptosis. However, the Notch signalling pathway did not influence the generation of CD8+ Tm cells. Most interestingly, IFN- regulation by the Notch signalling pathway depends on the activation context. Indeed, following Lm infection, lack of Notch receptors does not impact IFN- secretion by Te cells while it is significantly decreased following a DC immunisation suggesting a context dependant role for the Notch signalling pathway. Our findings provide a better understanding of the key signals provided by APC as well as the Notch signalling pathway, and thus the molecular mechanisms leading to CD8+ lymphocyte effector and memory generation which is crucial as this knowledge may ultimately lead to improved vaccination.