483 research outputs found
The role of the notch signalling pathway in vascular smooth muscle cell apoptosis
No full textThe Notch signalling system is a highly conserved method of cell-to-cell communication involved in cell fate decisions in many cell types. Until recently, the importance of the Notch signalling pathway was recognized in the embryonic but not in the adult vasculature. This project has successfully identified the presence of components of this pathway in adult vascular smooth muscle cells (VSMC). The effect of this pathway on serum deprivation- and cyclic strain-induced VSMC apoptosis has also been determined. This study reports that over-expression of components of the Notch signalling pathway results in a decrease in serum deprivation-induced VSMC apoptosis, whereas endogenous inhibition of the pathway increases apoptosis in these cells. The effect of the Notch signalling pathway on VSMC apoptosis is mediated, at least in part, in a CBF-1-dependent manner. A possible mechanism of the Notch signalling pathway regulation of apoptosis is through interaction with members of the Bcl-2 family of apoptotic proteins. Notch over-expression decreases pro-apoptotic bax, and increases anti-apoptotic bcl-xi expression in VSMC in a CBF-1-dependent manner. In addition, this study has shown previously unreported interaction of the Notch and NFkB signalling pathways in VSMC. Additionally, the effect of cyclic strain, a biomechanical force increased in many vascular disease states, was determined on components of the Notch signalling pathway and also on VSMC apoptosis. Increased cyclic strain results in decreased Notch signalling pathway component expression, and increased apoptosis. Over-expression of the Notch receptor, Notch 3, attenuates the cyclic strain-induced apoptosis in a bcl-xi-caspase 3-dependent manner. In vivo validation of the effects of altered biomechanical forces was performed through analysis of carotid artery, and hepatic portal vein, ligation models. In correlation with the in vitro results obtained, increased alterations in cyclic strain results in an inverse relationship between the Notch signalling pathway and apoptosis in the vessel. These findings may be relevant in the future management of vascular diseases
An investigation into how the cell cycle and the Notch signalling pathway regulate pronephrogenesis in Xenopus laevis
No full textThe 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
tricolor
No full textDiplacus tricolor (Hartw. ex Lindl.) NesomTricolor monkeyflowerMimulus tricolor500 yds e of cherokee Rd. ca. 5.5 mi sw of Cherokeein open grassy areas between lava beds1225 feetMalacothrix californica, Micropus californicuslocally abundantCorolla magenta; faintly fragrant
The role of NOTCH2 gene in human malignant glial brain tumours
No full textBackground: Glioblastoma multiforme (GBM), astrocytoma (A) and oligodendroglioma (OG)
are the neoplasms of the glial lineage in the Central Nervous System (CNS). Among them, GBM
occurs at the highest frequency and shows the shortest patient median survival time of some 10
months as compared for instance to the survival time of OG of about 10 years. Genetically, OG
differs from GBM by the frequent combination of loss of heterozygosity (LOH) on chromosomes
1p and 19q, which is associated with more favourable prognosis in OG patients. However, the
clinical significance of LOH on 1p in other glioma subtypes remained unknown.
Methods and Results: We identified a subgroup of GBM with LOH on centromeric chromosome
1p together with longer survival. The minimally lost area(s) in both GBM and OG converged at
the NOTCH2 locus on 1p11 and positively correlated with prognosis in GBM as well as in OG
patients. Comparison between gene expression of NOTCH2 and the genetic status at the NOTCH2
locus on chromosome 1p11 supported the hypothesis of a loss of function alteration of NOTCH2
in tumours. However, many GBMs do not display deletions at the NOTCH2 locus on 1p11 and do
express the NOTCH2 gene. Abundant expression of components of canonical NOTCH signaling
in these tumors and a positive correlation between NOTCH2 transcripts with the target gene
HES-1 (P=0.0001) indicated that functional NOTCH signaling in glioma is mainly driven by
NOTCH2. In addition, we defined TNC, the gene for the cell migration factor tenascin-C as a
novel target gene for NOTCH signaling. We further showed that activation of NOTCH signaling
was indeed promoting TNC-dependent glioma cell motility. Thus, together with the ability to
increase proliferation, canonical Notch signaling turned out to be critical for glioma progression.
We also found that non-canonical Notch signaling was associated with the maintenance of
tumorigenic potential of the GBM cells in soft agar culture. In addition, Notch2 had a prosurvival
effect on GBM cells by upregulating anti-apoptotic proteins Bcl-2 and Mcl-1,
independently of the canonical pathway. Finally, defective degradation pathway of Notch
receptors in GBM cells led to slow receptor turnover, thereby providing additional contribution to
the oncogenic function of Notch2.
Conclusion: This study identified aberrant multi-facetted oncogenic behaviours of Notch
proteins, in particular of Notch2, in GBM. This provided a molecular basis for the higher
aggressiveness of Notch2-positive GBM compared to Notch2-negative GBM or OG, and
suggested Notch2 as a sensible target for new therapeutic approaches against GBM
Iterative Role of Notch Signaling in Spinal Motor Neuron Diversification
No full textSummaryThe motor neuron progenitor domain in the ventral spinal cord gives rise to multiple subtypes of motor neurons and glial cells. Here, we examine whether progenitors found in this domain are multipotent and which signals contribute to their cell-type-specific differentiation. Using an in vitro neural differentiation model, we demonstrate that motor neuron progenitor differentiation is iteratively controlled by Notch signaling. First, Notch controls the timing of motor neuron genesis by repressing Neurogenin 2 (Ngn2) and maintaining Olig2-positive progenitors in a proliferative state. Second, in an Ngn2-independent manner, Notch contributes to the specification of median versus hypaxial motor column identity and lateral versus medial divisional identity of limb-innervating motor neurons. Thus, motor neuron progenitors are multipotent, and their diversification is controlled by Notch signaling that iteratively increases cellular diversity arising from a single neural progenitor domain
Patterning by cell-to-cell communication
No full textThis thesis addresses the question of how patterning may arise through cell-to-cell communication.
It combines quantitative data analysis with computational techniques to understand biological
patterning processes. The fi�rst section describes an investigation into the robustness of an evolved
arti�ficial patterning system. Cellular automata rules were implemented sequentially according to
the instructions in a simple `genome'. In this way, a set of target patterns could be evolved using
a genetic algorithm. The patterning systems were tested for robustness by perturbing cell states
during their development. This exposed how certain types of patterning rule had very di�fferent
levels of robustness to perturbations. Rules that generated patterns with complex divergent patterns
were more likely to amplify the e�ffect of a perturbation. When smaller genomes, comprising less
individual rules, were evolved to match certain target patterns, these were shown to be more likely
to select complex patterning rules. As a result, the developmental systems based on smaller genomes
were less robust than those with larger genome sizes.
Section two provides an analysis of the patterning of microchaetes in the epithelial layer of the
notum of Drosophila flies. It is shown that the pattern spacing is not sufficiently described by a
model of lateral inhibition through Delta-Notch signalling between adjacent cells. A computational
model is used to demonstrate the viability of long range signalling through a dynamic network of
�filopodia, observed in the basal layer of the epithelium. In-vivo experiments con�rm that when fi�lopodia lengths are effected by mutations the pattern spacing reduces in accordance with the model.
In the fi�nal section the behaviour of simple asynchronous cellular automata are analysed. It
is shown how these diff�er to the synchronous cellular automata used in the fi�rst section. A set of
rules are identifi�ed whose emergent behaviour is similar to the lateral inhibition patterning process
established by the Delta-Notch signalling system. Among these rules a particular subset are found
to produce patterns that adjust their spacing, over the course of their development, towards a more
ordered and densely packed state. A re-examination of the Delta-Notch signalling model reveals
that this type of packing optimisation could take place with either dynamic �filopodial signalling,
or as an alternative, transient Delta signalling at each cell. Under certain parameter regimes the
patterns become more densely packed over time, whilst maintaining a minimum zone of inhibition
around each Delta expressing cell. The asynchronous CA are also used to demonstrate how stripes
can be formed by cell-to-cell signalling and optimised, under certain conditions, so that they align
in a single direction. This is presented as a possible novel alternative to the reaction-di�ffusion
mechanism that is commonly used to model the patterning of spots and stripes
Effective Notch Stress Concept Investigations for Mode-III Loading & Response Conditions
No full textFatigue strength is typically a governing limit state for marine structures. Predicting an accurate fatigue lifetime is important for structures (e.g. wind turbines) that are supposed to be out at sea for decades. It is valuable for all type of structures that encounter stochastic sea loadings to optimise the design so that fatigue failure is predicted for, or material is saved in preventing over conservative estimates. This research contributes to the investigation of multiaxial fatigue concepts by getting insight in the mode-III loading & response conditions for the effective notch stress concept. The mode-III loading & response will in this research be represented by a tubular welded joint loaded under torsion. A semi-analytical formulation is developed to describe the weld notch shear stress distribution analytically. This formulation can be used as input for the effective notch stress concept. The formulation is based on three components, the notch stress, weld load carrying stress and far field stress component. Furthermore, the static force and moment equilibrium is satisfied. The weld load carrying stress component is based on a weld load carrying stress estimate. This coefficient is used because the weld geometry causes a local change in stiffness, a shift in neutral axis, meaning the weld becomes load carrying up to some extent. The distribution must be corrected for this phenomenon which is done by this estimated term. The geometry is a tubular welded joint. This specimen can be loaded with torsion which in the case of the tubular joint cause mode-III shear in the through thickness direction at the weld notch level. The weld load carrying stress estimate is based on the geometry ratios of the assessed specimens. It is individually fitted for all 6,300 geometries by comparing the results to stress distribution obtained with 2D finite element analysis. The results of the individual fitted load carrying stress estimates are used as input of a multi variable polynomial regression analysis. The regression analysis led to a polynomial function that describe the load carrying stress estimate which uses geometry ratios as input. The far field information and geometry dimensions are the input for the semi-analytical formulation to obtain the weld notch shear stress distributions analytically. Accurate results are obtained and therefore the formulation can be used as input for the effective notch stress concept. The effective notch stress concept is also based on a material characteristic length, ρ*, over which the stress distribution can be integrated to obtain the effective notch stress. This parameter is obtained using a log likelihood regression analysis and led to a value of ~1 [mm]. Although this is a good result, the confidence regarding the value is low due to low variety in the experimental data set and little data in general. The findings of this study will be used in further research regarding the 4D fatigue project and the effective notch stress concept in general at the Delft University of Technology.4D-FatigueMarine Technolog
Colon stem cell characterization in normal and tumoral tissues: description of a novel feed-forward circuit of Msi-1 regulation
No full textNormal tissues are organized in a hierarchical fashion, where rare somatic cells endowed with stem-like properties give origin to a population of differentiated cells forming the bulk of tissue. These self-maintaining cells, called stem cells, are characterized by 4 fundamental properties: longevity, multipotency, quiescence, and asymmetric cell division.
We decided to develop a new in vitro system for separation of stem-like epithelial cell based not on surface marker-driven selection, but by simply taking advantage of their slow proliferation rate in the absence of serum.
Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKHpos population survived in culture and formed spheroids; this population included subsets with slow (PKHhigh) and rapid (PKHlow) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr5 in PKHhigh cells; by cytofluorimetric analysis, Msi-1+/Lgr5+ cells were only found within PKHhigh cells, whereas Msi-1+/Lgr5- cells were also observed in the PKHlow population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1+/Lgr5+ cells. While CK20 expression was mainly found in PKHlow and PKHneg cells, a small PKHhigh subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin-1, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define a hierarchy of cellular subsets at different maturation stages: PKHhigh/Lgr5+/Msi-1+/CK20-, PKHhigh/Lgr5-/Msi-1+/CK20+, PKHlow/Lgr5-/Msi-1+/Ck20-, and PKHlow/ Lgr5-/Msi-1-/ CK20+ cells.
We next addressed the issue of cancer stem cells in colorectal cancer (CRC). Isolated cells from five primary colon tumors and 15 CRC metastatic samples were stained with PKH26 and cultured in PhEMA-treated plate in the absence of serum. As in normal samples, in tumors we identified a slowly cycling population able to persist in vitro in serum-free condition. On the contrary, in metastatic samples we could not document an in vitro selection of a PKHhigh population; actually PKHlow cells seemed to remain constant over several weeks. Cytofluorimetric analysis revealed that, at variance with normal samples, in metastatic samples Lgr5+/Msi-1+ cells were not only present in the slowly cycling PKHhigh population but also in PKHlow cells. No Lgr5 or Msi-1 expression was recorded in PKHneg cells. Both PKHhigh and PKHlow cells were able to form spheroids; in the presence of 10% FCS in uncoated plates they adhered to the plate and differentiated assuming an epithelial morphology. They eventually lost Msi-1 and Lgr5 expression after 14 days of culture in the presence of serum and adhesion conditions, and they also acquired CK20 expression.
Finally, we investigated the possible involvement of Notch signaling on regulation of Msi-1 levels in the metastatic cell line MICOL-14tum and primary metastatic samples. Seventy two hours of treatment with the Notch ligand DLL4 increased Msi-1 mRNA and protein levels. This phenomenon was largely prevented by the addition of an antibody neutralizing Notch2/3, whereas no reduction was detected after incubation with anti-Notch1. Since Msi-1 regulates Numb levels, its reduction led to increased Numb protein, as demonstrated by Western Blot analysis of MICOL-14tum and primary metastatic cells. Furthermore, after four days of treatment, formation of spheroids was significantly reduced by incubation with anti-Notch2/3 antibody in primary samples as well as in MICOL-14tum cells.
In conclusion, our data showed the possibility of deriving in vitro from normal colon tissue, without any selection strategy, an array of cellular subsets differentially expressing the most common stemness and differentiation markers of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in an anatomical position compatible with stem-like cells.
Furthermore in metastatic cell line and primary samples we described a novel feed-forward circuit involving Msi-1, Numb, Notch3 and Notch1. In particular, we demonstrated that Msi-1 could be considered a target of Notch3, and Notch3 could act as a positive regulator of Notch1 levels through Msi-1/Numb circuit
Mode-III fatigue of welded joints in steel maritime structures: Weld notch shear stress distributions and effective notch stress based resistance
No full textThe predominant mode-I response of maritime structures can be multiaxial, involving out-of-plane mode-III shear components. Semi-analytical mode-III notch stress distribution formulations have been established for critical details like welded T-joints and cruciform joints, reflecting (non-)symmetry with respect to half the plate thickness. Using a stress distribution formulation based effective notch stress as fatigue strength criterion, the mode-III welded joint mid-cycle fatigue resistance characteristics have been investigated. In comparison to mode-I, the material characteristic length and resistance curve slope estimate suggest the fatigue damage process to be even more an initiation related near-surface phenomenon. Mean shear stress effects seem insignificant.Ship Hydromechanics and Structure
Comparison between defects and micro-notches in multiaxial fatigue – The size effect and the gradient effect
No full textLien vers la version éditeur: http://www.sciencedirect.com/science/article/pii/S0142112308002156This paper attempts to improve the understanding of the multiaxial high cycle fatigue response of micro sized stress concentrations or notches of different geometries. The investigation is composed of an experimental part and a numerical part. In the former, three types of micro-notches or “artificial defects” are compared: spherical, elliptical and circumferential. All types have the same basic dimensions, the difference being the 3D geometry. The notches were machined on the surface of smooth cylindrical specimens made of mild steel. The fatigue limits under reversed tension (push–pull) and reversed torsional loading conditions for different micro-notch sizes have been experimentally determined. In the numerical part, finite elements simulations using a cyclic elasto-plastic material behaviour law show that the mechanical state ahead of the different stress concentrations change drastically with the loading mode and the geometry of the artificial defect. From a fatigue point of view, it is shown that a stress gradient correction is required for all the loading, size and geometry configurations. Once the gradient correction is made and a proper multiaxial criterion is used, it appears that the size effect due to increasing the loaded surface area at the notch tip for the different geometries is negligible compared to the gradient effect
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