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Functional Analysis of the Caenorhabditis elegans HP1 Homolog HPL-2 in a Chromatin ContextMiller, Elizabeth Victoria 09 1900 (has links)
The heterochromatin 1 (HP1) family of non-histone chromosomal proteins is
evolutionarily conserved and is involved in numerous biological processes, including the
stabilization of heterochromatin, a state of compacted DNA along a protein scaffold. HP1
proteins and trimethylated histone H3 on lysine 9 (H3K9me3) are major constituents of
heterochromatin and have been characterized extensively in vitro. The binding of HP1
proteins to H3K9 methylation marks plays an essential role in mammalian development
and chromatin organization. However, due to their critical function, dissecting the
molecular mechanism by which HP1 proteins exert their function in vivo is difficult. C.
elegans is a unique model because not only are deletion mutants of the two HP1
homologs, HPL-1 and HPL-2, viable, but also H3K9 methylation is not essential to worm
development. Interestingly, HPL-2 is alternatively spliced to generate two HP1 proteins,
but in vivo experimentation has vastly ignored the potential contributions of the
alternative transcripts to hpl-2 function, thus obfuscating which phenotypes associated
with hpl-2 knockdown are due to the loss of one or more of the splicing variants. In this
dissertation, I characterized the HPL-2 splicing variants (A and B) on a biochemical level in
relation to the canonical human HP1b protein and on a physiological level in splicing
variant-specific knockout worms. I show that both recombinant HPL-2A and HPL-2B bind H3K9me3 through their chromodomain (CD). But while HPL-2A acts as a canonical HP1
protein, namely it dimerizes and phase-separates like hHP1b, HPL-2B does not. In contrast
to recombinant protein, in extracts both proteins rely on other factors, such as the MBT
domain-containing protein LIN-61, for their recruitment to H3K9me3. Although HPL-2A
and HPL-2B display distinct characteristics in vitro, both hpl-2a and hpl-2b worms are
phenotypically wildtype. In agreement, knockout of either splicing variant leads to
upregulated expression of the other one, suggesting a certain level of functional
redundancy. Nevertheless, I show that the C-terminal extension of HPL-2B, which is
absent in HPL-2A, resembles that of the CEC-4 heterochromatin anchor. I therefore
hypothesize that the main functions of HPL-2 are distinct: HPL-2A mediates chromatin
compaction and HPL-2B facilitates heterochromatin anchoring to the nuclear periphery.
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Mouvements transmembranaires et effet sécrétagogue de l'albumine au niveau du syncytiotrophoblaste humain / Transmembrane movements and secretory effect of albumin at the human syncytiotrophoblast levelLambot, Nathalie 17 February 2006 (has links)
Le placenta assure les échanges materno-fœtaux et possède une fonction endocrine autonome. Les hormones placentaire lactogène (hPL) et chorionique gonadotrope (hCG) sont synthétisées par le syncytiotrophoblaste. A ce jour, les mécanismes impliqués dans le contrôle de la sécrétion de ces deux hormones ne sont pas connus. In vitro, l’influx d’ions Ca2+ entraîne une augmentation immédiate et soutenue de la libération d’hPL et d’hCG à partir d’explants de placentas à terme. En outre, l’élévation de la concentration extracellulaire en albumine, principale protéine maternelle circulante en contact direct avec le trophoblaste, stimule de manière immédiate et transitoire la libération d’hPL et d’hCG.
L’objectif de nos travaux a été de vérifier la spécificité de l’activité sécrétagogue de l’albumine au niveau du placenta, de caractériser les messagers cellulaires potentiellement impliqués dans la libération d’hPL et d’hCG, et de définir l’interaction entre l’albumine et le trophoblaste, en utilisant des explants provenant de placentas humains à terme.
Nos travaux démontrent que la riposte sécrétoire à l’albumine (5%, m/v) est largement mimée par d’autres agents colloïdaux (dextran et polygéline). Cette stimulation colloïdale de la libération d’hPL et d’hCG impliquerait une mobilisation de Ca2+ à partir de réserves intracellulaires. L’intervention de 3 messagers cellulaires a été envisagée: les IPs/DAG, l’AMPc, et le GMPc. Le fluorure de sodium, la forskoline, ou le nitroprussiate sodique, activateurs connus de la production respective des IPs, de l’AMPc, et du GMPc, augmentent de manière significative les taux placentaires de chacun de ces messagers, sans toutefois affecter la libération d’hPL ou d’hCG. De plus, l’élévation de la concentration extracellulaire en albumine (5%, m/v) ne modifie pas les taux des IPs, de l’AMPc et du GMPc dans les explants placentaires, tandis qu’elle stimule la sécrétion hormonale. Ces systèmes de signalisation, bien que fonctionnels au niveau du trophoblaste, ne joueraient donc pas un rôle majeur dans la régulation de la libération d’hPL et d’hCG.
Nos résultats mettent en évidence une internalisation rapide d’albumine marquée, avec de l’125I ou de la fluorescéïne, dans le syncytiotrophoblaste. Une large fraction de cette albumine est recyclée, intacte, vers la circulation maternelle selon un processus sensible à l’abaissement de la température et indépendant du cytosquelette. L’albumine marquée restant dans les explants placentaires est partiellement dégradée. Trois mécanismes ont été envisagés pour expliquer ces mouvements d’entrée et de sortie de l’albumine au sein du placenta humain: l’endocytose médiée par l’albondine via les caveolae, le système des coated pits clathrine-dépendant, et l’endocytose médiée par la mégaline. Par immunohistochimie, nous avons montré que, dans le tissu placentaire, la caveoline-1, protéine caractéristique des caveolae, est localisée uniquement dans l’endothelium des capillaires fœtaux. La clathrine, au niveau des coated pits, et la mégaline se trouvent au contraire dans le syncytiotrophoblaste. La méthyl-b-cyclodextrine et l’hydrochlorure de chlorpromazine, inhibiteurs d’une endocytose dépendant de la clathrine, réduisent significativement l’internalisation placentaire de l’albumine marquée. Par contre, le DIDS ou le NPPB, susceptibles de perturber l’endocytose médiée par la mégaline, n’affectent pas la captation d’albumine marquée par les explants placentaires. L’albumine pénétrerait donc dans le syncytiotrophoblaste principalement par un processus clathrine-dépendant. La mégaline ne jouerait ici qu’un rôle mineur dans l’entrée de la protéine. Un tel processus de recyclage de l’albumine pourrait être similaire à celui décrit pour les immunoglobulines G au niveau du syncytiotrophoblaste.
Ces mouvement d’entrée et de sortie de l’albumine ne semblent pas associés à la stimulation de la libération d’hPL et d’hCG par l’albumine. Ils pourraient par contre participer significativement, étant donné leur ampleur, à la nutrition fœtale. L’albumine est en effet un transporteur notoire d’ions et d’acides gras, molécules qui pourraient être acheminées au fœtus via le phénomène de recyclage placentaire de l’albumine mis en évidence par ce travail. /
The human placenta is the site of all maternal-fetal exchanges, and is also an active endocrine organ. Placental lactogen (hPL) and chorionic gonadotrophin (hCG) hormones are synthesized by the syncytiotrophoblast. So far, the mechanisms involved in the regulation of both hormones secretion remain elusive. In vitro, calcium inflow causes an immediate and sustained rise in the hPL and hCG releases from human term placenta explants. Moreover, increasing the extracellular concentration of albumin, the major maternal plasma protein in direct contact with the human trophoblast, stimulates the hPL and hCG releases in an immediate and transient way.
Our study have aimed to check the specificity of this secretory effect of albumin, to investigate the potential cellular messengers involved in the hPL and hCG releases, and to define the interaction between albumin and the throphoblast layer, using human term placenta explants.
Our results indicate that the triggering effect of albumin (5%, w/v) is largely mimicked by two other colloidal agents (dextran and polygelin). This “colloidal” stimulation of the hPL and hCG releases would involve the mobilization of calcium from intracellular pools. Three cellular messengers have been considered to mediate this process: the IPs/DAG, the cAMP, and the cGMP. Sodium fluoride, forskolin, or sodium nitroprusside, known activators of respectively the IPs, cAMP, and cGMP production, significantly increase the placental content of each of those messengers, without modifying the hPL and hCG releases. In addition, raising the extracellular concentration of albumin does not cause any change in the placental level of IPs, cAMP, and cGMP, while stimulating the hormonal release. These three signaling pathways are thus functional in human term trophoblast but do not appear to significantly modulate the hPL and hCG secretions.
Our findings show that albumin, labeled with 125I or with fluorescein, is rapidly internalized into the syncytiotrophoblast. Thereafter, the intact protein is largely recycled to the maternal circulation, through a temperature-sensitive and cytoskeleton-independent process. The labeled albumin remaining in placental explants is partially degraded. Three different mechanisms could participate to the albumin entry into the human placenta: the albondin-mediated endocytosis via the caveolae, the clathrin-dependent coated pits system, and the megalin-mediated endocytosis. Using immunohistochemistry, caveolin-1, marker of the caveolae, is localized in the endothelium of the fetal capillaries and not in the syncytiotrophoblast. By contrast, clathrin and megalin are observed only in the syncytiotrophoblast. Methyl-b-cyclodextrin, and chlorpromazine hydrochloride, known inhibitors of the clathrin-dependent endocytotic process, significantly reduce the placental uptake of labeled albumin. On the other hand, DIDS or NPPB, able to perturb the megalin-mediated endocytosis, do not affect the labeled albumin uptake. Thus, albumin seems to be internalized into the syncytiotrophoblast mainly through a clathrin-dependent mechanism. Megalin would only play a minor role in this process. Such movements of albumin in the human placenta may be similar to the recycling process reported for IgG at that site.
The placental apical recycling of albumin is not associated to the albumin triggering effect on the hPL and hCG releases. This quantitatively significant internalization process may participate to the fetus’ nutrition. Indeed, Albumin carries ions and fatty acid, which could be brought to the fetus via the protein recycling evidenced by our study.
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Regulation of binding of HP1 associated complexes to chromatin and their role in transcription regulation in C. elegans vulva developmentOstwal, Yogesh 21 October 2105 (has links)
No description available.
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Základní škola / Elementary SchoolOsička, Martin January 2020 (has links)
The theme of the diploma thesis is the preparation of project documentation for the construction of two designed buildings of the Primary School. Building plot for the planned new building is located near the existing nursery school near the center of Vracov. The campus is designed to provide basic education in a total of 18 classes with a proposed total capacity of 540 pupils. The complex consists of 4 main buildings that functionally divide individual operations. These are designed as free-standing, SO01 and SO04 objects will be two-storey, without basement. SO02 and SO03 objects as four-storey without basement. The construction system is designed as a prefabricated reinforced concrete skeleton with filling masonry from ceramic blocks to thin-walled masonry mortar. The ceiling construction consists of pre-stressed ceiling panels SPIROLL in combination with reinforced concrete. The staircases connecting the floors are designed in reinforced concrete. Vertical communication between floors will also be made possible by barrier-free passenger lifts. The roof cladding consists of a single-shell construction with thermal insulation made of EPS, the covering will be made of PVC foil weighted with a layer of peacock. The building will be insulated with a façade thermal insulation system with a ventilated gap and external surface treatment of HPL façade cladding panels. Paved pedestrian roads will be made of concrete interlocking pavement. The road for vehicle traffic will be with an asphalt surface. Parking spaces are designed from drainage concrete pavement. The project documentation was processed in ArchiCAD.
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Školka - Praha / Nursery building - PrahaMiheličová, Gabriela January 2019 (has links)
The aim of my thesis is to design and elaborate the project documentation for construction of the nursery school in the cadastral area Prague-Černý most. It is a two-storey partially basement building, witch with its layout corresponds to the conditons of the building operation. Nusery school is designed for 4 departments after 24 kids. The construction system of the object is designed as Porotherm system. External load-bearing structures are designed as ventilated facade with thermal insulation of 180 mm thickness. Vertical load-bearing structures of 1st and 2nd floor will be lined with brick blocks Porotherm 38 profi and 1.S bricks from the Best 30 permanent formwork. The inner bearing walls are designed with Porotherm 24 profi and Porotherm 11,5 Profi is used for non-load-bearing masonry. The exterior facade cladding is designed from HPL compact laminate boards. The internal staircase is half-turn stairs, reinforced concrete, monolithic. Horizontal supporting structures consist of Porotherm ceramic concrete system (POT beams and miako insert) with a thickness of 250 mm. The roof is designed as a vegetation, some of witch is supplemented with a gutter. The windows and doors are plastic-aluminium. Floor coverings are designed taking into the use of individual rooms such as floor screed, ceramic tiles and cork floors. The main aim of my work was to solve the disposition for the given purpose, to design a suitable construction system and to draw up the drawing documentation including the text part.
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Řešení pro clusterování serverů / Server clustering techniquesČech, Martin January 2009 (has links)
The work is given an analysis of Open Source Software (further referred as OSS), which allows use and create computer clusters. It explored the issue of clustering and construction of clusters. All installations, configuration and cluster management have been done on the operating system GNU / Linux. Presented OSS makes possible to compile a storage cluster, cluster with load distribution, cluster with high availability and computing cluster. Different types of benchmarks was theoretically analyzed, and practically used for measuring cluster’s performance. Results were compared with others, eg. the TOP500 list of the best clusters available online. Practical part of the work deals with comparing performance computing clusters. With several tens of computational nodes has been established cluster, where was installed package OpenMPI, which allows parallelization of calculations. Subsequently, tests were performed with the High Performance Linpack, which by calculation of linear equations provides total performance. Influence of the parallelization to algorithm PEA was also tested. To present practical usability, cluster has been tested by program John the Ripper, which serves to cracking users passwords. The work shall include the quantity of graphs clarifying the function and mainly showing the achieved results.
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Analýza procesu výroby HPL desek použitých na stavby dětských hřišť společností Kompan a.s / Analysis of the production process of HPL panels applied for playgrounds made by Kompan IncVik, Michal January 2014 (has links)
The theoretical part consists of the introduction of company KOMPAN A.S. In addition, there are also introduced the main parts of production, especially CNC workshop, shop floor workplace and warehouse. In the experimental part there is introduced HPL material first. The following is a description of the testing including used tools and materials provided by Kompan Czech Republic s.r.o. Testing was focused on measurement of force load of instruments using piezoelectric dynamometer. The obtained data was processed and analyzed. The last part of the master's thesis is economic evaluation of the production of selected product, which consist of HPL panels. In the following discussion there are evaluate obtained results. In the conclusion part are summarized main results.
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Proliferation and Differentiation of Intestinal Caco-2 Cells Are Maintained in Culture with Human Platelet Lysate Instead of Fetal Calf SerumWanes, Dalanda, Naim, Hassan Y., Dengler, Franziska 03 May 2023 (has links)
Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS.
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Mouvements transmembranaires et effet sécrétagogue de l'albumine au niveau du syncytiotrophopblaste humain / Transmembrane movements and secretory effect of albumin at the human syncytiotrophoblast levelLambot, Nathalie 17 February 2006 (has links)
Le placenta assure les échanges materno-fœtaux et possède une fonction endocrine autonome. Les hormones placentaire lactogène (hPL) et chorionique gonadotrope (hCG) sont synthétisées par le syncytiotrophoblaste. A ce jour, les mécanismes impliqués dans le contrôle de la sécrétion de ces deux hormones ne sont pas connus. In vitro, l’influx d’ions Ca2+ entraîne une augmentation immédiate et soutenue de la libération d’hPL et d’hCG à partir d’explants de placentas à terme. En outre, l’élévation de la concentration extracellulaire en albumine, principale protéine maternelle circulante en contact direct avec le trophoblaste, stimule de manière immédiate et transitoire la libération d’hPL et d’hCG.<p><p>L’objectif de nos travaux a été de vérifier la spécificité de l’activité sécrétagogue de l’albumine au niveau du placenta, de caractériser les messagers cellulaires potentiellement impliqués dans la libération d’hPL et d’hCG, et de définir l’interaction entre l’albumine et le trophoblaste, en utilisant des explants provenant de placentas humains à terme.<p> <p>Nos travaux démontrent que la riposte sécrétoire à l’albumine (5%, m/v) est largement mimée par d’autres agents colloïdaux (dextran et polygéline). Cette stimulation colloïdale de la libération d’hPL et d’hCG impliquerait une mobilisation de Ca2+ à partir de réserves intracellulaires. L’intervention de 3 messagers cellulaires a été envisagée: les IPs/DAG, l’AMPc, et le GMPc. Le fluorure de sodium, la forskoline, ou le nitroprussiate sodique, activateurs connus de la production respective des IPs, de l’AMPc, et du GMPc, augmentent de manière significative les taux placentaires de chacun de ces messagers, sans toutefois affecter la libération d’hPL ou d’hCG. De plus, l’élévation de la concentration extracellulaire en albumine (5%, m/v) ne modifie pas les taux des IPs, de l’AMPc et du GMPc dans les explants placentaires, tandis qu’elle stimule la sécrétion hormonale. Ces systèmes de signalisation, bien que fonctionnels au niveau du trophoblaste, ne joueraient donc pas un rôle majeur dans la régulation de la libération d’hPL et d’hCG. <p><p>Nos résultats mettent en évidence une internalisation rapide d’albumine marquée, avec de l’125I ou de la fluorescéïne, dans le syncytiotrophoblaste. Une large fraction de cette albumine est recyclée, intacte, vers la circulation maternelle selon un processus sensible à l’abaissement de la température et indépendant du cytosquelette. L’albumine marquée restant dans les explants placentaires est partiellement dégradée. Trois mécanismes ont été envisagés pour expliquer ces mouvements d’entrée et de sortie de l’albumine au sein du placenta humain: l’endocytose médiée par l’albondine via les caveolae, le système des coated pits clathrine-dépendant, et l’endocytose médiée par la mégaline. Par immunohistochimie, nous avons montré que, dans le tissu placentaire, la caveoline-1, protéine caractéristique des caveolae, est localisée uniquement dans l’endothelium des capillaires fœtaux. La clathrine, au niveau des coated pits, et la mégaline se trouvent au contraire dans le syncytiotrophoblaste. La méthyl-b-cyclodextrine et l’hydrochlorure de chlorpromazine, inhibiteurs d’une endocytose dépendant de la clathrine, réduisent significativement l’internalisation placentaire de l’albumine marquée. Par contre, le DIDS ou le NPPB, susceptibles de perturber l’endocytose médiée par la mégaline, n’affectent pas la captation d’albumine marquée par les explants placentaires. L’albumine pénétrerait donc dans le syncytiotrophoblaste principalement par un processus clathrine-dépendant. La mégaline ne jouerait ici qu’un rôle mineur dans l’entrée de la protéine. Un tel processus de recyclage de l’albumine pourrait être similaire à celui décrit pour les immunoglobulines G au niveau du syncytiotrophoblaste.<p><p>Ces mouvement d’entrée et de sortie de l’albumine ne semblent pas associés à la stimulation de la libération d’hPL et d’hCG par l’albumine. Ils pourraient par contre participer significativement, étant donné leur ampleur, à la nutrition fœtale. L’albumine est en effet un transporteur notoire d’ions et d’acides gras, molécules qui pourraient être acheminées au fœtus via le phénomène de recyclage placentaire de l’albumine mis en évidence par ce travail. /<p><p>The human placenta is the site of all maternal-fetal exchanges, and is also an active endocrine organ. Placental lactogen (hPL) and chorionic gonadotrophin (hCG) hormones are synthesized by the syncytiotrophoblast. So far, the mechanisms involved in the regulation of both hormones secretion remain elusive. In vitro, calcium inflow causes an immediate and sustained rise in the hPL and hCG releases from human term placenta explants. Moreover, increasing the extracellular concentration of albumin, the major maternal plasma protein in direct contact with the human trophoblast, stimulates the hPL and hCG releases in an immediate and transient way.<p><p>Our study have aimed to check the specificity of this secretory effect of albumin, to investigate the potential cellular messengers involved in the hPL and hCG releases, and to define the interaction between albumin and the throphoblast layer, using human term placenta explants.<p><p>Our results indicate that the triggering effect of albumin (5%, w/v) is largely mimicked by two other colloidal agents (dextran and polygelin). This “colloidal” stimulation of the hPL and hCG releases would involve the mobilization of calcium from intracellular pools. Three cellular messengers have been considered to mediate this process: the IPs/DAG, the cAMP, and the cGMP. Sodium fluoride, forskolin, or sodium nitroprusside, known activators of respectively the IPs, cAMP, and cGMP production, significantly increase the placental content of each of those messengers, without modifying the hPL and hCG releases. In addition, raising the extracellular concentration of albumin does not cause any change in the placental level of IPs, cAMP, and cGMP, while stimulating the hormonal release. These three signaling pathways are thus functional in human term trophoblast but do not appear to significantly modulate the hPL and hCG secretions. <p><p>Our findings show that albumin, labeled with 125I or with fluorescein, is rapidly internalized into the syncytiotrophoblast. Thereafter, the intact protein is largely recycled to the maternal circulation, through a temperature-sensitive and cytoskeleton-independent process. The labeled albumin remaining in placental explants is partially degraded. Three different mechanisms could participate to the albumin entry into the human placenta: the albondin-mediated endocytosis via the caveolae, the clathrin-dependent coated pits system, and the megalin-mediated endocytosis. Using immunohistochemistry, caveolin-1, marker of the caveolae, is localized in the endothelium of the fetal capillaries and not in the syncytiotrophoblast. By contrast, clathrin and megalin are observed only in the syncytiotrophoblast. Methyl-b-cyclodextrin, and chlorpromazine hydrochloride, known inhibitors of the clathrin-dependent endocytotic process, significantly reduce the placental uptake of labeled albumin. On the other hand, DIDS or NPPB, able to perturb the megalin-mediated endocytosis, do not affect the labeled albumin uptake. Thus, albumin seems to be internalized into the syncytiotrophoblast mainly through a clathrin-dependent mechanism. Megalin would only play a minor role in this process. Such movements of albumin in the human placenta may be similar to the recycling process reported for IgG at that site.<p><p>The placental apical recycling of albumin is not associated to the albumin triggering effect on the hPL and hCG releases. This quantitatively significant internalization process may participate to the fetus’ nutrition. Indeed, Albumin carries ions and fatty acid, which could be brought to the fetus via the protein recycling evidenced by our study.<p><p><p> <p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished
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Optimizing Applications and Message-Passing Libraries for the QPACE ArchitectureWunderlich, Simon 18 July 2012 (has links) (PDF)
The goal of the QPACE project is to build a novel cost-efficient massive parallel supercomputer optimized for LQCD (Lattice Quantum Chromodynamics) applications. Unlike previous projects which use custom ASICs, this is accomplished by using the general purpose multi-core CPU PowerXCell 8i processor tightly coupled with a custom network processor implemented on a modern FPGA. The heterogeneous architecture of the PowerXCell 8i processor and its core-independent OS-bypassing access to the custom network hardware and application-oriented 3D torus topology pose interesting challenges for the implementation of the applications. This work will describe and evaluate the implementation possibilities of message passing APIs: the more general MPI, and the more QCD-oriented QMP, and their performance in PPE centric or SPE centric scenarios. These results will then be employed to optimize HPL for the QPACE architecture. Finally, the developed approaches and concepts will be briefly discussed regarding their applicability to heterogeneous node/network architectures as is the case in the "High-speed Network Interface with Collective Operation Support for Cell BE (NICOLL)" project.
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