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No description available.

The fall in circulating oestrogen (E2) after the menopause leads to an increased rate of bone remodelling, excessive osteoclast activity and a greater fracture risk. Until recently hormone replacement therapy (HRT) was prescribed to post-menopausal women to prevent bone loss, however HRT is associated with an elevated incidence of cardiovascular disease, stroke and cancer. These side-effects led to an interest in naturally occurring compounds with oestrogenic action such as phytoestrogens (PEs), which are non-steroidal-plant derived compounds. Human trials and animal studies suggest a beneficial effect of PEs on bone mass, although their ability to modify osteoclast formation in response to key inflammatory cytokines has not been examined. The aim of the following studies was to determine the effect of physiologically relevant concentrations of genistein, coumestrol and daidzein on TNF-α-induced osteoclast formation, osteoblasts differentiation and T cell activity. Genistein (10-7 M), daidzein (10-5 M), and coumestrol (10-7 M) significantly reduced TNF-α-induced TRAP positive osteoclast formation and bone resorption, which was prevented by the E2 antagonist ICI 182,780. The suppressive action on osteoclast formation was associated with a significant reduction in TNF-α-induced c-fos and NFATc1 mRNA expression and NFATc1 nuclear translocation. Constitutive c-fos expression prevented the inhibitory action of PEs on osteoclast differentiation, resorption and NFATc1 expression. The effect of PEs, in the presence or absence of the anabolic nutritional factor zinc, on osteoblasts differentiation and bone nodule formation was examined in-vitro. Coumestrol (10-5 to 10-7 M), daidzein (10-5 to 10-6 M) and genistein (10-5 M) enhanced bone nodule formation and ALP activity in human osteoblasts, and this effect was significantly augmented in the presence of zinc (10-5 M). Furthermore, PEs and zinc increased Runx2 mRNA expression and Zn2+ augmented the inhibitory effect of PEs on RANKL/OPG ratio. This suggests that in addition to the direct inhibitory effect on osteoclast formation PEs also in-directly reduce the osteoblastsic stimulus for osteoclast formation and promote bone formation. E2 deficiency is thought to promote osteoclastogenesis by modifying Thelper1 (Th1) cell proliferation and inflammatory cytokine production in particular TNF-α. I therefore examined the effect of PEs on T cell proliferation and inflammatory cytokine production. All PEs prevented the augmentative effect of con A stimulated T cells on osteoclast formation in co-culture. However the mechanism of action varied, genistein reduced con A stimulated TNF-α, IL-1β and RANKL expression with little effect on viability, coumestrol decreased cell viability and TNF-α expression whereas the inhibitory effect of daidzein was mediated via suppression of viable T cell number. This study provides novel evidence that PEs have multiple effects on bone cell activity, directly inhibiting TNF-α-induced osteoclast formation, reducing the osteoblasts and T cell derived stimulus for osteoclast formation and augmenting osteoblasts differentiation and bone formation. Thus, PEs have a potential role in the treatment of post-menopausal osteoporosis and inflammatory skeletal disorders and that the beneficial effect noted in previous studies is mediated through multiple mechanisms.

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo. They exhibit unlimited proliferation in culture and have the ability to differentiate into all three germ layers of the developing organism, a property defined as pluripotency. Previously it was reported that growth factor-bound protein 2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that harbours the pluripotent founder cells of the foetus. GRB2 is an adapter protein involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in response to extracellular signals. It has also been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in response to fibroblast growth factor (FGF) signaling. The work presented in this thesis examines the role of Grb2 in ES cells and describes previously unreported contributions of this adaptor protein in regulating ES cell growth and differentiation. It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow relatively normally in ES growth medium containing serum. However, in serum free conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA mini gene. Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog expression was uniformly upregulated, in contrast to the heterogeneous pattern reported in serum-based medium. Colony expansion on the substratum appears to be compromised, although there is no apparent defect in the initial attachment of Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in serum free medium could be due to lengthening of the G1 phase of the ES cell cycle. In an attempt to identify the signalling deficiency responsible for the growth defect of Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated component of the MAPK pathway that acts downstream of Grb2 in the MAPK pathway. Although both approaches increased MAPK signalling they were unable to rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive. It is hypothesised that under these conditions cells undergo hyper-self-renewal at the cost of growth. Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6, MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically and immunohistochemically identifiable neuronal cells confirmed differentiation of ectodermal cell types, indicating Grb2 is not required for neuronal differentiation. However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation pathway. This may be due to a defect in GATA factor expression since these factors are essential for cardiogenesis. In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells. Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to prevent this differentiation. However, biochemical analysis of the cells indicates that this occurs when ERK activation is very low, indicating differentiation was not MAPK-independent. Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is dispensable for differentiation in to some lineages. However as differentiation of Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.

Supply Chain Management (SCM) aims to synchronize the requirements of customers with the flow of materials from suppliers, in order to satisfy the needs of the customers as costefficiently as possible. This has become a difficult task due to several developments in the market, such as increased competition, increased demand variability, increased product variety, increased amounts of customer-specific products, and shortening product life cycles. These developments, due in part to globalization, provide additional management challenges and new practices in which supply chains are designed and managed, and can eventually make the difference between companies staying competitive or not. The overall purpose of this thesis is to investigate how complexity and globalization affect supply chain design and operations. The main emphasis has been on producing descriptive results of the studied phenomenon. This research involves five case studies covering international transportation structures used in SCM, the selection of supply chain strategies in different business environments, and the role of information systems and technology in achieving the objective of SCM. In this thesis it has been concluded that in order to cope with increasingly complex and fragmented markets companies need more differentiated transportation structures, modes, and supply chains. Furthermore, to effectively manage this, information systems and advanced decision support tools are required. In addition, this thesis has shown that current taxonomies for supply chain strategy selection are too simplistic due to three major problems: they mediate that it is a question of choosing one supply chain strategy for the entire company, they regard markets as rather homogeneous, and they link each supply chain strategy to a specific business context. Instead, it has been concluded that in order to better satisfy differing customer needs in various markets it is increasingly necessary to develop a differentiated supply chain strategy by utilizing different manufacturing and delivery strategies concurrently. Thus, a need exists for new taxonomies for supply chain strategy selection which recognize that the markets are becoming more fragmented and complex, that customer preferences differ across customer/market segments, and that there is a need to differentiate the supply chain strategy. This thesis also highlights several requirements of a differentiated supply chain strategy. Firstly, extended supply chain collaboration is required, since a differentiated supply chain strategy will involve more supply chain partners than a traditional supply chain strategy. Secondly, there is a need for more transportation mode alternatives, particularly intermodal, both in supply and distribution operations, due to the fact that differentiation requires diversity. In this thesis, intermodal landbridge freight services are highlighted as one interesting avenue, which could potentially facilitate a more differentiated supply chain strategy. Thirdly, more integrated information systems are needed along with decision support tools. This study illustrates that agent based modeling appears to be an interesting method for developing realistic decision support tools in the context of complex supply chains. An interesting aspect for further research is to investigate how different manufacturing and delivery strategies can be used concurrently in international supply chains. Moreover, there are several requirements and opportunities of a differentiated supply chain strategy, and these have to be investigated further

No description available.

Neuroblastoma is the most common pediatric cancer in infants, arising from the sympathoadrenal lineage of the neural crest. Despite recent advances in other pediatric cancers, long term survival in high risk cases of neuroblastoma remains below 40%. Therefore, to develop successful therapeutics targeting high risk tumors, further research into the mechanisms involved in high risk tumor formation is necessary. Prognosis in neuroblastoma is determined by a number of factors, including certain genetic and biological variables. The genetic variable correlated most with high risk disease is amplification of the MYCN gene, which is present in ~25% of tumors. Additionally, ~70% of these MYCN-amplified tumors express the neurotrophin receptor TrkB, and its ligand, brain-derived neurotrophic factor (BDNF), with concurrent expression of these proteins correlated with high risk disease independent of MYCN-amplification status. To better understand factors influencing MYCN-amplified tumor cell phenotype, and the role of TrkB signaling in high risk neuroblastoma, the experiments in this dissertation examined growth factor effects on MYCN-amplified tumor cells from the TH-MYCN mouse model of neuroblastoma, as well as the creation, and expression of a constitutively active TrkB receptor in a neural crest derived cell line. Overexpression of MYCN targeted to the sympathoadrenal lineage by the tyrosine hydroxylase (TH) promoter is sufficient to cause neuroblastoma in 100% of mice homozygous for the transgene (TH-MYCN mice). Screening growth factors, in vitro treatment of tumor cells from dissociated TH-MYCN tumors with ciliary neurotrophic factor (CNTF) was found to promote differentiation marked by increased neurite outgrowth, and withdrawal of actively dividing cells from the cell cycle. These effects were both concentration dependent, and specific to CNTF, as all other neurotrophic factors tested had no effect on differentiation. Furthermore, TH-MYCN tumor cells were found to highly express the receptor for CNTF, CNTFR both in vitro and in vivo. Testing the ability of CNTF to affect tumor growth in vivo, CNTF treatment attenuated subcutaneous tumor growth of the TH-MYCN tumor-derived cell line NHO1S in wild type 129/SvJ mice. Therefore, CNTF signaling may be a potential therapeutic target in MYCN-amplified neuroblastoma. In addition to being significantly correlated with a poor prognosis in neuroblastoma, the presence of activated TrkB signaling promotes a more aggressive phenotype in established neuroblastoma cell lines. However, whether TrkB signaling is sufficient to transform neural crest derived cells had not been established. To determine the role of TrkB signaling in malignant transformation, the two immunoglobulin-like (Ig) ligand binding domains were removed from a full length rat TrkB receptor. Expression of this receptor, termed IgTrkB, leads to elevated phosphorylated Erk levels in the absence of ligand, indicating the receptor is constitutively active. When expressed in the neural crest derived cell line NCM-1, constitutive TrkB signaling confers a highly transformed phenotype characterized by enhanced proliferation, anchorage-independent cell growth, anoikis resistance, and matrix invasion. Furthermore, IgTrkB NCM-1 cells upregulate transcripts for a number of cancer promoting genes, in addition to the poor prognosis marker MYCN. In vivo, IgTrkB NCM-1 cells form highly aggressive tumors, requiring euthanasia of mice by 15 days following injection, while wild type cells fail to grow. Thus, TrkB signaling is sufficient to transform cells derived from the neural crest.

Abstract An Analysis of Specialized Reading Instruction in High School English Classes for Students with Disabilities Included In General Education By Kathy Rosvold Beasley, M.Ed. A dissertation submitted in partial in fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2010. Major Director: Dr. Whitney H. Sherman Associate Professor, School of Education This study is a qualitative case study that examined and analyzed the instructional strategies implemented by high school English teachers when teaching reading to students with disabilities in inclusive classrooms. Ten teachers who teach high school English on collaborative teams made up of a general and a special educator participated in the study. The participants taught at a comprehensive high school that is one of ten high schools in a large school division. Two observations of each team were carried out to examine how teachers differentiate instruction. Team interviews were conducted to gain information about how teachers use data on student readiness, interests, and learning profiles to plan, implement, and assess the learning of their students. More students with disabilities are receiving their education in inclusive settings so that they have greater access to the general education curriculum in the least restrictive environment. Differentiating instruction is being implemented by teachers so that they can meet the unique needs of their students. Results of previous studies have found that students have had more opportunities for success on general education expectations when teachers implemented of elements of differentiation. These studies focused on examining single elements of differentiation including student readiness, interests, learning profiles, content, process, or products. This study focused on how teachers plan for, implement, and assess their students by implementing the differentiation framework in inclusive high school English classes. The results of this study suggest that teaching teams made up of a general and special educator can work collaboratively together to provide instruction to all of their students including those with disabilities. Teachers use data to determine their students’ levels of readiness, interest, and learning profiles to design lessons that meet the unique needs of their learners. The participants planned their instruction in their collaborative English classes using student readiness, interest, and learning profile data, but emphasized the beneficial aspects of planning instruction based on students’ interest. Teachers said that students were highly motivated to participate in class and complete assignments when activities where based upon student interest. The study’s results also indicate that the role of building level administrators was vital in the collaborative process. The participants discussed how the principal’s leadership paved the way for teachers to access data on their students and allowed for teams to have joint planning periods, focused special educators on teaching one content area of instruction, provided opportunities for teams to have input in their longevity, and encouraged teacher participation in personnel decisions regarding changes in team membership.

This final thesis gives an overview of possibilities given to Member states by European law to differentiate the legislature the way it fits their national needs. It might be difficult for all Member states to participate in all European policies as it is stated in Treaties, directives or regulations as the Member state cannot or does not want to follow the mainstream of integration. European law then provides the means how to differentiate from the provisions to secure that any harm will be caused to a Member state. In the first part of the thesis, some basic terms to understand the subject are introduced such as differentiation, harmonisation, flexibility and integration itself. In the second chapter you can find the process of harmonization and the power within that the institutions of European Union can operate. Some disputes about how the powers are exercised are also introduced here. The differentiated integration had some development which laid the foundations to the current situation and therefore the development is described in this chapter. There are more ways how to differentiate within the European law, chapter number 3 gives a basic overview of these possibilities. It mentions multiple-speed Europe, variable geometry and integration á la carte, and gives some practical examples to understand...

La biogenèse du ribosome est un processus indispensable à la prolifération cellulaire car elle permet la synthèse protéique assurant la croissance avant la division cellulaire. Les ribosomopathies telles que le syndrome myélodysplasique 5q- et l’anémie de Blackfan-Diamond sont dues respectivement à une mutation d’un gène codant une protéine ribosomique (RP) et à l’haploinsuffisance en RPS14, RP de la petite sous-unité du ribosome. Les patients atteints de l’une de ces ribosomopathies présentent un défaut de l’érythropoïèse suggérant que celle-ci est particulièrement dépendante du ribosome. L’érythropoïèse est le processus qui permet la formation de globules rouges à partir de cellules souches hématopoïétiques et consiste en différents stades de différenciation appelés érythroblastes. C’est dans ce contexte que je me suis intéressée au ribosome au cours de l’érythropoïèse. Dans un premier temps, nous avons caractérisé la biogenèse du ribosome dans des cellules érythroïdes primaires humaines et murines. Pour cela nous avons adapté une technique de SILAC pulsé et mis au point la ribomique, technique de protéomique permettant l’analyse de la biogenèse du ribosome dans des échantillons de cellules primaires basée sur l’identification presque exhaustive des protéines ribosomiques. À l’aide de la ribomique et par d’autres techniques, nous avons mis en évidence une diminution de la biogenèse du ribosome après le stade érythroblaste basophile. Nous avons également montré que cette biogenèse du ribosome est en partie sous le contrôle de la voie mTORC1 régulée par les deux cytokines fondamentales de l’érythropoïèse : le Stem Cell Factor (SCF) et l’érythropoïétine (EPO). L’expression par l’érythroblaste des récepteurs des deux cytokines permet une biogenèse du ribosome optimale. L’inhibition de la biogenèse du ribosome par le CX-5461, inhibiteur spécifique de l’ARN polymérase I, ou par la rapamycine, inhibiteur de mTORC1, entraîne une accélération de la différenciation érythroïde soulignant un rôle de la biogenèse du ribosome au cours de l’érythropoïèse. L’inhibition de la voie mTORC1 modifie l’ordre de clivage de l’ARNr, reflet d’une modification de sa maturation. Les expériences de ribomique dans les érythroblastes humains ont également permis de mettre en évidence la présence de paralogues de RP et la sous-représentation de certaines RPs au sein des ribosomes suggérant une hétérogénéité des ribosomes dans les érythroblastes humains. Parallèlement, un modèle mimant le syndrome 5q- a été développé par une approche shRPS14 dans une lignée humaine érythroleucémique dépendante de l’EPO. L’inhibition de RPS14 entraîne un défaut de biogenèse de la sous-unité 40S du ribosome aboutissant à une diminution des ribosomes entiers formés et une diminution de la traduction globale. Cependant une traduction est maintenue. Le défaut de biogenèse de la sous-unité 40S entraîne une augmentation de la quantité de c-KIT, récepteur du SCF et une diminution de la quantité de GATA1, facteur de transcription spécifique de l’érythropoïèse. Nous avons mis en évidence que la diminution de GATA1 est due à une diminution de sa traduction tandis que la traduction d’autres protéines est conservée dans ce contexte d’altération de la biogenèse du ribosome. Nous avons ensuite réalisé une analyse des transcrits présents dans les fractions polysomales correspondants à la traduction la plus efficace. Nous avons montré grâce à ce traductome que les propriétés thermodynamiques des parties 5’ et 3’UTR des ARNm modulent leur traduction dans le contexte d’inhibition de RPS14. Ces données suggèrent que l’altération de la biogenèse du ribosome peut aboutir à une modification du programme traductionnel. Ce travail montre que la biogenèse du ribosome diminue au cours de l’érythropoïèse et participe à la différenciation érythroïde. La voie mTORC1 participe au contrôle de cette biogenèse. / Ribosome biogenesis is a key event allowing cell growth before division. Defective RB recognized in ribosomopathyinherited Diamond-Blackfan anemia and 5q- syndrom. In this study, we aimed at investigating the regulatory role of RB during the erythroid precursor maturation which is characterized by a cell size reduction during 2 to 3 rapid cell divisions. We used two in vitro systemsé of expansion and differentiation of erythroblasts (E.) derived of immature hematopoietic progenitors from human mobilized peripheral blood or mouse fetal liver. The expansion step is supported by the Stem Cell Factor (SCF) and the second step depends on erythropoietin (EPO). The structure of the nucleolus was studied by electron microscopy. Compared to immature proerythroblasts (proE), a dramatic size reduction and change in nucleolar structure (ie. the disappearance of fibrillar and dense fibrillar components) is observed at the stage of mature polychromatophilic E. suggesting a loss of functionality. RB was measured by a pulsed SILAC (Stable Isotopic Labeling by Amino acids in Culture cell) proteomic assay that quantified the incorporation of newly synthesized ribosomal proteins in the ribosome. Both in mouse and human models, immature proE expanded upon SCF and EPO demonstrate a maximal RB with a renewal rate of 60% and 50% every 14h and 24h, respectively. By contrast, RB rapidly interrupted with the disappearance of proE and basophilic E after the switch to EPO alone. Consistently, the quantities of ribosomal RNA (rRNA) 45S precursor estimated by qPCR are maximal in proE and almost null in orthochromatophilic E. Inhibition of RB at proE stage by RNApol I specific inhibitor (CX-5461) accelerates the onset of terminal erythroid differentiation suggesting that RB is a rate limiting factor for final maturation. We then hypothesize that degree of signaling intensity in response to SCF and EPO may control the level of RB. To address this question, we investigated the mTORC1 (mechanistic Target Of Rapamycin Complex 1) pathway which is directly involved in RB through its substrate p70S6Kinase. Activation of P-p70S6Kinase and P-Rps6, as well as ribosome renewal, are twice more elevated in response to SCF and EPO than to EPO alone. Furthermore, inhibition of mTORC1/p70S6K/Rps6 pathway by rapamycin disrupts RB and leads to an acceleration of terminal erythroid differentiation.This study demonstrates that the collapse of RB promotes erythroid cell terminal maturation and shows the regulatory role of mTORC1 pathway on RB during erythropoiesis.