La cellule principale de l’os sous-chondral est l’ostéoblaste qui joue un rôle central dans la production qualitative et quantitative de la matrice ostéoïde. Plusieurs études montrent que le collagène de type I et la phosphatase alcaline sont des marqueurs précoces de la différenciation des ostéoblastes tandis que l’ostéocalcine (OCN) et la minéralisation sont des marqueurs des stades tardifs de cette différenciation. L’os sous-chondral est le site actif de nombreux changements morphologiques qui peuvent être différents au cours de l’arthrose (OA) mais qui sont partie prenante du processus pathologique. Ces changements consistent en une formation de la matrice osseuse importante associée une inhibition de la minéralisation. Ces phénomènes peuvent être reliés aux changements phénotypiques des ostéoblastes. La galectine-3 (gal-3) est un facteur inflammatoire qui a été détecté dans le tissu synovial et dans le liquide synovial lors d’inflammation d’OA. Des études antérieures ont montré que la gal-3 participait à la destruction du cartilage et inhibait fortement la production d’OCN par les ostéoblastes OA. Ces faits ont permis de suggérer que la gal-3 pouvait participer soit à l’initiation soit à la progression de l’arthrose. Peu d’études ont globalement été réalisées sur le rôle de la galectine-3 dans l’arthrose et encore moins sur le rôle de gal-3 sur les altérations de l’os sous-chondral.Dans ce contexte, ce travail de thèse a consisté à caractériser les ostéoblastes arthrosiques, puis investigué la modulation du phénotype des ostéoblastes arthrosiques par gal-3 en et enfin identifier les mécanismes cellulaires impliqués. D’une part, nous avons identifié deux populations ostéoblastes OA grâce à l’expression d’OCN. Dans les conditions basales, ces deux populations expriment de façon différentielle le TGF-ß1, Wnt5b et DKK2, ce qui suggère une différenciation et un phénotype hétérogènes des ostéoblastes chez les patients OA. D’autre part, nous confirmons le rôle délétère de la gal-3 dans l’articulation lors d’inflammation puisqu’elle stimule la production de collagénase 1 impliquée dans la dégradation osseuse. De plus, elle accentue la perturbation phénotypique des ostéoblastes qui produisent plus de leptine lors d’épisodes hypoxiques. Bien que plusieurs ligands membranaires puissent médier les effets de gal-3, 4F2hc semble jouer un rôle récurrent / Osteoblasts are the main cells in subchondral bone (SCB), which are responsible for the bone matrix production. Their differentiation can be evaluated by type I collagen and alkaline phosphatase in the early stage and by osteocalcin (OCN) and mineralization in the late stage. Alterations of SCB are essential episodes of osteoarthritis (OA) and are represented by a significant bone formation accompanied with abnormal hypomineralization. These changes in SCB are related to phenotypic modifications of osteoblasts. Galectin-3 (Gal-3) is an inflammatory factor markedly detected in the synovial tissue and synovial fluid during OA inflammation. Previous studies have demonstrated that gal-3 was deleterious for cartilage and inhibited the production of OCN in OA osteoblasts. These findings suggest that gal-3 can participate in either the initiation or progression of osteoarthritis. So far, a few studies have been conducted to explore the role of Gal-3 in OA and particularly related to SCB. In this context, the thesis has consisted to characterize OA osteoblasts, to investigate the modulation of the OA osteoblast phenotype by gal-3 and finally to identify the involved cellular mechanisms. We have identified two populations of OA osteoblasts according to the OCN expression. Under basal conditions, these two populations express TGF-ß1, Wnt5b and DKK2 differentially, suggesting various differentiation and heterogeneous phenotype of osteoblasts in OA patients. Moreover, we confirmed the deleterious role of gal-3 in the joint during inflammation since it stimulates the production of collagenase 1 involved in bone degradation. In addition, it emphasizes the disruption of phenotypic osteoblasts by producing more leptin during hypoxic episodes. Although several membrane ligands can mediate the effects of gal-3, 4F2hc seems to play a prominent role

Studien har haft som syfte att undersöka hur lärare i årskurs 1-3 planerar och genomförundervisning i teknik. Frågeställningarna som tagits fram för att för att få svar på studiens syfteär: hur beskriver lärarna att de planerar för teknikämnet, allmänt och i relation till det centralainnehållet för årskurs 1-3?, hur beskriver lärarna att de undervisar relaterat till det centralainnehållet i teknik för årskurs 1-3? samt vilka undervisningsmetoder används, enligt lärarna?Dessa frågor har det sökts svar på genom kvalitativa intervjuer med åtta lärare i årskurs 1-3.I resultatet framkommer att teknik är ett ämne som inte fullt ut har fått fäste i årskurs 1-3, då detvisar sig att flera lärare i studien saknar förtrogenheten med ämnets kursplan och planering ochundervisning beskrivs till stor del bedrivas tillsammans med No-ämnena. Teknikämnet har haften egen kursplan i tjugo år. Trots detta saknas på många skolor lärare med utbildning i tekniksamt material. Det framgår dock också i resultatet att några av de intervjuade lärarna ärintresserade och väl insatt i ämnets kursplan, även material i form av Skellefte-tekniken och NTAanvänds av flertalet av lärarna i studien. Teknikämnet behöver stärkas ytterligare som eget ämneoch i högre utsträckning undervisas separat för att ge eleverna en god uppfattning av vad som är teknik

Plusieurs études ont suggéré une implication des glycogène synthase kinases 3 (GSK3) dans la carcinogenèse, notamment du pancréas. Des études ont rapporté des résultats contradictoires quant à l’impact des GSK3 sur la survie cellulaire. Au niveau du pancréas, il a été observé que l’inhibition des GSK3 inhibe la croissance entre autres via la régulation de la voie JNK ou NFkB. Les inhibiteurs des GSK3 sont présentement à l’étude comme traitement de différentes pathologies, notamment pour le cancer pancréatique. Une meilleure compréhension des voies de signalisation régulées par les GSK3 sera donc nécessaire. Nous avons entrepris ces travaux afin de mieux comprendre les mécanismes impliqués dans la régulation de la survie des cellules pancréatiques tumorales par les GSK3. Nous avons démontré que l’inhibition des GSK3 induit l’apoptose et l’autophagie dans les cellules pancréatiques tumorales humaines. L’inhibition des GSK3 stimule l’autophagie autant dans les cellules pancréatiques tumorales que non tumorales, alors que l’apoptose est induite spécifiquement dans les cellules tumorales. Contrairement à l’apoptose, l’autophagie est induite indépendamment de la voie JNK-cJUN suite à l’inhibition des GSK3. Nos résultats démontrent que l’inhibition des GSK3 mène à l’inactivation de la voie mTORC1 qui pourrait contribuer à l’induction de l’autophagie. D’autre part, nos travaux ont démontré pour la première fois que les GSK3 régulent le facteur de transcription EB (TFEB) dans les cellules pancréatiques tumorales. En effet, l’inhibition des GSK3 entraîne la déphosphorylation de TFEB, notamment sur la Ser211, la dissociation des 14-3- 3 et sa translocation nucléaire. Nos résultats suggèrent que la régulation de TFEB par les GSK3 impliquerait des Ser/Thr phosphatases et pourrait être indépendante de l’activité mTORC1. L’inhibition de l’autophagie ou la déplétion de l’expression de TFEB sensibilise les cellules pancréatiques tumorales à l’apoptose induite suite à l’inhibition des GSK3 suggérant un rôle pro-survie de l’autophagie et de TFEB dans ces cellules. Enfin, l’inhibition des GSK3 semble mener à l’inhibition de la glycolyse qui contribuerait à l’induction de l’apoptose. En résumé, nos résultats démontrent que l’inhibition des GSK3 induit à la fois des signaux pro-apoptotiques et pro-survie suggérant que l’équilibre entre ces signaux dicterait l’impact des GSK3 sur la survie des cellules pancréatiques tumorales humaines.

The aim of this diploma thesis was to study the role of posttranslational modifications of phosducin and their role in the interaction with the 14-3-3 protein as well as the influence of the complex formation on these modifications. Phosducin is a 33kDa protein commonly present in photoreceptor cells of the retina as well as other tissues. Despite many experiments, its physiological functions are still elusive. It has been speculated that fosducin plays an important regulatory role in visual phototransduction pathway, regulation of blood pressure and expression of G-proteins. The phosducin function is regulated through binding to the 14-3-3 protein, a regulatory protein involved in many biochemical processes. Phosducins binding to 14-3-3 protein requires phosphorylation of two serine residues Ser-54 and Ser-73 within the N-terminal domain of phosducin. However, the role of the 14-3-3 protein binding in the regulation of phosducin function is still unclear. In this diploma thesis proteins 14-3-3ζ∆C and phosducin (mutation Q52K) were successfully expressed and purified. The effect of the complex formation on phosducin posttranslational modifications was investigated using limited proteolysis and dephosphorylation. These experiments revealed that the complex formation significantly slowed down both...

Context. In the present network inventive world, there is a quick expansion of switches and protocols, which are used to cope up with the increase in customer requirement in the networking. With increasing demand for higher bandwidths and lower latency and to meet these requirements new network paths are introduced. To reduce network load in present switching network, development of new innovative switching is required. These required results can be achieved by Software Define Network or Traditional layer-3 technologies.Objectives. In this thesis, the end to end (e2e) transmission performance of OpenFlow and Layer-3 switches and their dynamic characteristics are investigated using network simulation.Methods. To replicate real life network topology and evaluate e2e transmission performance, a simulation based test-bed is implemented for both OpenFlow switch and layer-3 switch. The test beds are implemented using Network Simulator-3 (NS3). A two-tire network topology is designed with specified components for performance evaluation.Results. The performance metrics like throughput, average delay, simulation time and Packet Delivery Ratio (PDR) are measured, results are analyzed statistically and are compared. The behavior of network traffic in both the topologies are understood using NS-3 and explained further in the thesis.Conclusions. The analytical and statistical results from simulation show that OpenFlow switching performs relatively better than layer-3 switching.

Thesis advisor: James P. Morken / This dissertation describes the development of two enantioselective methods of carbon-carbon bond formation. Chapter one discusses the development of an enantioselective Pd(0)-catalyzed conjugate addition of allylboronic acid pinacol ester to α,β-unsaturated methylidene ketones. Utilizing the same rationale for regio- and enantiocontrol as in the related enantioselective conjugate allylation of arylidene ketones, this method addresses the gap in technology by expanding the scope of the reaction to include alkyl-substituted enones. Chapter two examines the coupling of allyl electrophiles and allyl metal reagents. With computational insight into the reaction mechanism, a catalyst system was designed to control regioselectivity and enantioselectivity. Isotope labeling studies were carried out to probe the mechanism of the transformation. The reaction also proves to be diastereoselective when a substituted allyl boron reagent is employed. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

CHARLES UNIVERSITY IN PRAGUE THE FACULTY OF NATURAL SCIENCE Department of Physical and Macromolecular Chemistry The summary of the doctoral thesis Study of interactions of forkhead transcription factor FOXO4 with DNA and the 14-3-3 protein RNDr. Petr Vácha Scientific supervisor: Prof. RNDr. Tomáš Obšil, Ph.D. Prague 2015 Abstract This doctoral thesis deals with the interaction of human forkhead transcription factor FOXO4 with DNA and regulating 14-3-3 protein respectively. The main aim of this work was detailed characterization of interaction between DNA binding domain of protein FOXO4 with two canonical DNA sequences and further clarifying the role of the 14-3-3 protein in the regulation of activity of protein FOXO4. FOXO transcription factors are potent activators of the transcription of genes, which affect a variety of cellular processes. FOXO4 protein belongs to the family of forkhead transcription factor, which is a group of several tens of proteins, whose common feature is a highly conserved DNA- binding domain. Summary of the DNA binding specificity of these proteins, namely what precisely determines the small differences in the binding properties of individual forkhead proteins, despite the large amount of available structural data remains still unclear. Therefore, detailed characterization of...

This dissertation is focused on 30 kDa protein phosducin (Pdc) and on the regulation of its function through the interaction with 28 kDa adaptor protein 14-3-3. These two proteins participate in G-protein signal transduction pathways, especially in the process of light signal transduction. It is assumed that Pdc binds to the Gtβγ complex of G-protein called transducin and through this interaction it inhibits the reassociation of Gtβγ with Gtα thus reducing the visual signal transfer. This process is thought to participate in a long- term light adaptation. The regulation of Pdc function is further regulated by its phosphorylation and subsequent binding to the 14-3-3 protein. It has been speculated that the 14-3-3 binding plays a key role in the inhibition of the interaction between phosphorylated Pdc (Pdc-PP) and Gtβγ. The formation of the 14-3-3/Pdc-PP complex leads to the reassociation of Gtβγ with Gtα and consequently to the amplification of visual signal transfer. Nevertheless, the mechanism by which the 14-3-3 protein binding inhibits the interaction between Pdc and Gtβγ remains elusive. The main aims of this dissertation were: (i) to investigate the structure of Pdc in its apo-state (in the absence of the binding partner) and in the complex with 14-3-3, and (ii) to suggest the mechanism of the...

EN Protein-protein interactions have an exceptional position among other mechanisms in the regulation of signal transduction. Their systematic investigation is very important and logical step in the process of understanding to the transduction and its mechanisms at a molecular level. During my Ph.D. I was particularly interested in three important processes. ASK1 kinase is well-known initiator of the apoptosis. Under physiological conditions it is maintained in an inactive state by its two interaction partners the 14-3-3 protein and TRX1. These two proteins dissociate in the presence of reactive oxygen species by unclear mechanism and the kinase is therefore activated. The next process is an interaction between the 14-3-3 protein and phosducin and investigation of their role in the G protein signalling especially important in the biochemistry of vision. The third process is an activation of protein Nth1 through the interaction with Bmh1, yeast analog of the 14-3-3 protein, and calcium cations. I employed various biophysical method, particularly analytical ultracentrifugation, in order to explain molecular mechanisms of described processes. These techniques were used to solve the low-resolution structures of complexes TRX1 and the 14-3-3 protein with corresponding binding domains of ASK1. These...

Tumores são definidos como microambientes compostos não só pelas células malignas, mas também por células endoteliais, fibroblastos e leucócitos, que promovem o crescimento tumoral e a angiogênese. Galectina-3, uma proteína que se liga a b- galactosídeos, é abundantemente expressa por monócitos/macrófagos, dentre outros leucócitos. Inúmeras evidências sugerem que galectina-3 atua como uma molécula reguladora da resposta inflamatória. Tendo em vista que o infiltrado inflamatório pode promover a progressão de tumores, o objetivo do presente trabalho foi avaliar se galectina-3, expressa tanto pela célula tumoral como pelas células estromais, modula o crescimento de melanoma. Para tal, células de melanoma murino Tm1 foram transfectadas com o gene de galectina-3. Ambos clones celulares (galectina-3 positivos e negativos) foram injetados na intrafáscia ou no subcutâneo de camundongos (fêmeas) C57BL/6 selvagens e/ou nocautes para o gene de galectina-3 para análise da implantabilidade e crescimento tumoral. Com relação à implantabilidade, não foi observado diferenças no estabelecimento de uma massa tumoral proliferativa em animais selvagens inoculados com células Tm1 transfectadas ou não com o gene de galectina-3 em animais selvagens. Em relação a taxa de crescimento dos tumores, nenhum animal nocaute inoculado com células Tm1 galectina-3 positivas apresentou tumores de dimensões mensuráveis até o 11º dia pós-inóculo. Independente do nível de expressão de galectina- 3 pela célula tumoral, os tumores originados nos animais nocautes apresentavam menor massa em gramas comparados ao grupo selvagem, sugerindo que galectina-3 expressa pelas células estromais promove o crescimento tumoral. Ainda, os tumores originados nos animais nocautes e no grupo selvagem inoculado com células Tm1 galectina-3 positivas apresentavam menor extensão de área necrótica do que os animais selvagens inoculados com células Tm1 galectina-3 negativas. Interessantemente, os animais selvagens e nocautes inoculados com células Tm1 galectina-3 positivas apresentaram tumores com menor área vascular e menor número de estruturas vasculares funcionais quando comparados aos animais selvagens inoculados com células Tm1 galectina-3 negativas. A análise de expressão gênica nos tumores mostrou que os níveis relativos de RNAm de VEFG (fator de crescimento de endotélio vascular) foram menores nos animais inoculados com células Tm1 galectina-3 positivas em relação aos inoculados com células Tm1 galectina-3 negativas, indicando que galectina-3 expressa pelas células tumorais atua como uma molécula anti-angiogênica. Finalizando, o presente trabalho sugere que galectina-3 pode atuar como uma molécula pró- ou anti-tumoral, dependendo do tipo celular que a expressa no microambiente tumoral. / Tumors have been described as microenvironments composed not only by malignant cells, but also by endothelial cells, fibroblasts and leukocytes, which can promote tumor growth and angiogenesis. Galectin-3, a b-galactoside binding protein, is expressed by monocytes/macrophages and others leukocytes. In fact, several lines of evidence suggest that galectin-3 act as master regulators of the inflammatory response. Based on the fact that the inflammatory infiltrate can promote tumor progression, the proposal of this study was to evaluate if galectin-3, either from tumor or stromal cells could modulate melanoma growth. Tm1 murine melanoma cell line was transfected with the galectin-3 gene. Both clones (galectin-3 negative and positive) were injected in the foot pad or subcutaneous in female C57BL/6 wild-type (WT) and galectin-3 knock-out (KO) mice to tumor engraftment and growth analysis. There was no difference in the tumor engraftment between animas injected with Tm1 galectin-3 positive or negative cells. In addition, any knock-out mice injected with galectin-3 positive cells had measurable tumors up to day 11 post inoculation. Regardless the galectin-3 expression level in the melanoma cell, tumors from galectin-3 KO mice were smaller than those from WT animals, suggesting that galectin-3 expressed by stromal cells promotes tumor growth. Moreover, tumor necrotic area was smaller in KO mice and in wild-type animals injected with Tm1 galectin-3 positive cells compared to wild type animals injected with Tm1 galectin-3 negative cells. Interestingly, both vascular area and the number of functional vessels in animals injected with galectin-3 positive Tm1 cells were smaller in WT as well as in KO mice compared to the same animals injected with galectin-3 negative Tm1 cells. Gene expression analysis showed that VEGF (vascular endothelial growth factor) mRNA levels were smaller in wild type animals injected with Tm1 galectin-3 positive cells compared to those injected with Tm1 galectin-3 negative cells, indicating that galectin-3 expressed by tumor cells can act as an anti-angiogenic molecule. The present study suggests that galectin-3 can act either as a pro or antitumoral molecule, depending on which type of cell (tumoral or stromal) this lectin is expressed within tumor microenvironment.