The regulation of gene expression resulting in the formation of the mammalian cerebral cortex is tightly regulated by a group of transcription factors. The deletion of any one of these transcription factors results in numerous defects whose nature and severity depends on the role of the transcription factor in the regulation of complex gene regulatory networks involved in development. There is currently relatively little knowledge about the gene networks that these transcription factors control and how they exert their regulatory effects. The paired-box transcription factor Pax6 has been identified as a master regulator of gene networks involved in cortical development and its deletion results in numerous cortical defects such as an abnormally thin cortical plate and a vastly expanded proliferative zone. Previous work in our lab identified a list of candidate genes that are likely to be regulated by Pax6 in the developing cortex. Members of the Notch signalling pathway were potential Pax6 targets of particular interest since Notch signalling plays a crucial role in the maintenance of neural progenitor cells during development and consequently plays a critical role during corticogenesis. Our work aims to identify the regulatory relationship between Pax6 and Notch ligands Dll1 and Jag1 during cortical development. Analysis by flow cytometry and double labelling analysis of both gene and protein expression has provided insight into the relationship between Pax6 and Dll1 in progenitor cell subpopulations during cortical development. In situ hybridisation and qPCR results confirmed that loss of Pax6 causes loss of Dll1 expressing cells and downregulation of Jag1, indicating that both ligands are regulated by Pax6. Bioinformatic screening and analysis by luciferase assay suggests that Jag1 is a likely candidate to be a direct target of Pax6.

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.

Adult stem cells are vital for tissue maintenance. Stem cell over proliferation results in tumour formation, whilst loss of stem cells causes tissue degeneration and a variety of diseases. Stem cell maintenance and proliferation is regulated through somatic structures called niches. The germline stem cell niche in Drosophila ovary has been well defined and it is useful to better understand the interactions between niche and stem cells. Notch signalling is needed for germline stem cell niche creation and maintenance. The aim of this thesis is to better understand both the regulation of Notch signalling during development and its requirement in the adult niche. The first paper, "Reversible regulation of stem cell niche size through dietary control of Notch signalling", revolves around the dynamicity of the niche. The niche is found to respond to diet stimuli and has the ability to be restored. Notch was previously found to be involved in the maintenance of the niche. We found that Notch signalling is altered by diet, and we dissect its different maintenance and recovery roles in the ovary. In the second paper, "ZO-1 controls stem cell niche assembly by acting as an upstream regulator of Deltex-dependent Notch signalling", we show how Notch signalling is finely regulated during niche formation through interplay with the proteins Polychaetoid and Deltex. This paper leads to a better understanding of how the niche is assembled and how Notch signalling is regulated in a context-dependent way. The obtained results from both papers will help understand the dynamics of the model germline stem cell niche, and how Notch signalling is found at the convergence between internal and external stimuli regulating the ovary's response to a changing environment.

Breast and pancreatic cancers are among the major causes of cancer mortality in our society. There has been a significant decline in mortality from breast cancer over the last two decades, while pancreatic cancer has an exceptionally poor prognosis. Although these malignancies have very different clinical outcomes they share the common feature that metastatic disease is almost uniformly fatal. The existence of cancer stem cells has been postulated as a major factor in tumour recurrence after traditional chemo- or radio-therapy. Addressing this important clinical question requires a deeper understanding of the biology of normal and cancer stem cells and the signalling pathways involved in their regulation. The identity of the pancreatic stem cell remains elusive. However, using a murine model of haematopoietic stem cell (HSC) transplantation I have demonstrated for the first time transdifferentiation of these bone marrow derived cells into mature pancreatic acinar cells, where they appear to contribute to cell turnover ultimately forming acini and lobules. These data show that HSC have surprising developmental plasticity and provide insight into a potential stem cell niche in the pancreas. The Hedgehog, Wnt and Notch signalling pathways play a critical role in early development and in the maintenance and self-renewal of stem cells. There is also increasing evidence that dysregulation of these pathways contributes to the development of many malignancies. There is relatively little information regarding their role in breast cancer development and progression. I used immunohistochemistry for key proteins in these pathways, sonic hedgehog, beta-catenin and Notch 1 in three substantial series of human breast lesions and determined that abnormal expression of these proteins is an early event in the development in breast cancer, and is associated with particular breast cancer subtypes, Shh and beta-catenin expression is associated predominantly with the basal-like phenotype and Notch 1 with the HER2 amplified phenotype. Overexpression of Shh in particular confers a worse clinical outcome in invasive ductal carcinoma. Furthermore, increased levels of Shh in a 3D culture model of non-transformed mammary epithelial cells resulted in disorganisation of acini and the development of an abnormal discohesive phenotype. Finally the role of Shh was investigated in a mammary epithelial transplantation model, where overexpression of Shh resulted in the development of hyperplasia of the mammary ductal epithelium. Together these data confirm that the Hedgehog, Wnt and Notch developmental pathways are dysregulated in breast cancer and represent viable targets for further investigation of potential novel therapies in breast cancer.

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Eco Log AB i Söderhamn är en tillverkare av skogsmaskiner. De tillverkar både skotare och skördare. På deras skördare sitter fyra pendelarmar som ersätter hjulupphängningen på fordonet. Dessa motverkar att fordonet lutar vid körning i sluttningar. Varje pendelarm drivs av en hydraulkolv som justerar nivelleringen. Då inga beräkningar med avseende på utmattning och livslängd genomförts sedan en omkonstruktion av pendelarmarna är det nödvändigt att analysera och utvärdera pendelarmarna. Syftet med examensarbetet är att analysera ett antal, maximalt tre, av pendelarmens svetsfogar med avsikt på utmattning. Jämförelse mellan de tre olika utvärderingsmetoderna Effective notch stress, Nominell spänning och Hot spot kommer genomföras. På begäran av Eco Log redovisas ej några värden på beräknade livslängder och spänningar utan endast en jämförelse mellan de olika metoderna redovisas där Nominella spännings-metoden används som referensvärde. En inledande spänningsanalys på en pendelarm på deras största skördare, Eco Log 590D, genomfördes. Denna analys, där endast en förenklad pendelarm användes, visade att det mest belastade svetsförbandet befann sig i nivå med infästningen av lagret i ytterlådans konstruktion. Detta svetsförband analyserades sedan vidare med avseende på utmattning. De mer noggranna utmattningsanalyserna baserades på tre olika utvärderingsmetoder för spänningar, Hot spot-, Effective notch stress- och Nominella spännings-metoden. Hot spot-metoden inkluderar membranspänningar och böjspänningar för det utvalda svetsförbandet. Effective notch stress inkluderar mebramspänningar, böjspänningar och den olinjära spänningstoppen för att kunna analysera spänningskoncentrationer i svetsrot eller tå. Nominella spänningar inkluderar normalspänningar. Genom att modellera det utvalda svetsförbandet med dessa tre metoder erhölls information om den maximala spänningsvidden. Denna spänningsvidd applicerades sedan i en arbetsgång för utmattningsberäkningar där en livslängd beräknades fram. Denna livslängd blev för Nominella och Effective notch-metoden blev densamma och för Hot spot-metoden blev den dubbelt så lång. Denna beräkningsgång baseras på International Institute of Welding.Denna beräknade livslängd var betydligt mindre än vad Eco Log hade förväntat sig. Dock är osäkerheten vid beräkningar och test med avseende på utmattning stor och därav borde även provning genomföras för att säkerställa livslängden. / Eco Log AB is a manufacturer of forestry equipment. They manufacture both forwarders and harvesters. On their harvesters there are four pendulum arms that replace the suspension on their vehicles. The pendulum arms anti tilts the vehicle when driving in slopes. Every pendulum arm is individually driven by hydraulic cylinders that adjust the leveling. Given that no calculations with respect to fatigue and service life since a redesign were made, analysis and evaluation of the pendulum arm was necessary. The purpose of this thesis is to analyze an number of, maximum three, of the pendulums welds with respect to fatigue. Comparison between three different evaluation methods Effective notch stress, Nominal stress and Hot spot will be conducted. On request from Eco Log no values regarding calculated service lifetime and stress will be presented, only a comparison between the different methods were the Nominal stress-method is used as reference. An initial stress analysis on the pendulum arm on their largest harvester, the Eco Log 590D, was conducted. This analysis, which only used a simplified pendulum arm, indicated that the most load carrying load were located at the attachment of the bearing thus on the outer box-constructuib. This welded joint was then further analyzed with respect to fatigue. These fatigue analysis was based on three different evaluation methods, Hot spot-, Effective notch stress- and Nominal stress-method. The Hot spot method includes membrane stress and bending stress for the selected weld. Effective notch stress includes membrane stress, bending stress and the nonlinear stress peak in order to analyze stress concentration in the weld root and weld toe. Nominell stress includes normal stresses. By constructing the selected welded joint with these three evaluation methods, information regarding the maximal stress range was given. This stress range was then applied to a specific working procedure for fatigue calculations were the results obtained in expected service-life. The service-life for the Nominal and Effective notch stress-method were almost identic meanwhile for the Hot spot-method the service-life was twice as long. This working procedure is based on the International Institute of Welding. This calculated service-life were significantly less than what Eco Log had expected. The uncertainty of calculation for fatigue is however big and therefore testing should also be carried out to ensure the service life of pendulum arm.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs.

The sensory cells of the inner ear, hair cells, provide vertebrates with the ability to detect auditory stimuli and balance. In mammals, cochlear hair cells, those responsible for hearing, do not regenerate. Zebrafish hair cells do regenerate. Gaining an understanding of the role and regulation of the genes involved in the formation and regeneration of these cells may provide information important for the development of genetic therapies. We show that zebrafish atoh1 acts as the proneural gene responsible for defining the equivalence group from which hair cells form. Expression of atoh1 is dependent upon Fgf and Pax. Atoh1 induces expression of delta, resulting in activation of Notch and subsequent lateral inhibition. Another factor known to be important for hair cell formation in mice is Sox2. In zebrafish, sox2 expression is downstream of Atoh1, Notch and Fgf. Zebrafish Sox2 is not required for hair cell formation, but rather Sox2 is important for hair cell maintenance. In zebrafish, otic hair cell regeneration has not yet been characterized. We show that, following laser ablation, hair cells regenerate by way of transdifferentiation. We further show that this regeneration requires Sox2 activity. These data suggest that Sox2 acts to maintain support cell plasticity. This role is likely conserved because Sox2 is also important for stem cell plasticity in mammals. This new understanding of the role and regulation of both Atoh1 and Sox2 provides essential information that can be used to further efforts to provide genetic therapies for hair cell regeneration in mammals.

Thèse de doctorat : Médecine. Biologie. Immunologie : Nantes : 2007. / Bibliogr.

Head and neck cancer is diagnosed in more than 50,000 Americans each year, resulting in roughly 11,000 deaths. For this disease, a typical therapeutic regimen involves cisplatin, a radiosensitizer, given alongside targeted irradiation. While technological advances such as IMRT have been useful in sparing normal tissues from radiotherapy, the salivary glands occupy much of the head and neck and surround several lymph nodes, and thus, non-diseased salivary glands are often damaged. This causes reduced salivary output, damaged oral mucosa, dysphagia, malnutrition and tooth decay. Often, these side-effects are so severe that patients discontinue treatment, however, in many cases, salivary gland damage is permanent, and treatment options are palliative. Specifically, muscarinic-cholinergic agonists are used to enhance secretion from remaining salivary cells, although due to non-specific action, these drugs have a number of ill-effects. It is clear that therapies are needed to prevent radiation-induced salivary gland damage, as well as to restore glandular function in patients who are already suffering.Previous work from our group has shown that salivary gland dysfunction results from loss of acinar cells to radiation-induced apoptosis. Importantly, a single intravenous dose of IGF1 can prevent apoptosis and preserve salivary output when given immediately prior to irradiation. Because of its broad effects, however, IGF1 may never be a viable clinical option. Instead, our goal is to identify signaling events that mediate the radioprotective effects of IGF1 downstream of Akt. Because radiation-induced apoptosis in salivary glands is p53-dependent, we assessed the contributions of the p53 homologs p63 and p73 to the DNA damage response. Here, we show that IGF1 enhances cell cycle arrest following irradiation by reducing inhibitory binding of deltaNp63 to the p21 promoter. We hypothesize that IGF1-induced cell cycle arrest may allow time for DNA repair, thus preventing apoptosis and maintaining salivary function. In addition, we indicate chronic signaling events downstream of p63 that may contribute to permanent loss of salivary function by blocking differentiation of salivary progenitor cells. Together, these results indicate that p63 may be a valid therapeutic target for both prevention of damage and restoration of function in irradiated salivary glands.