• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 3
  • 1
  • Tagged with
  • 6
  • 3
  • 3
  • 2
  • 2
  • 2
  • 2
  • 2
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Nuclear decondensation in Xenopus extracts

Philpott, Anna January 1991 (has links)
No description available.
2

CONTROLLING PLATELET SECRETION TO MODULATE HEMOSTASIS AND THROMBOSIS

Joshi, Smita 01 January 2018 (has links)
Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent occlusive thrombosis without triggering bleeding. My work addresses this question by showing how the rates and extents of granule secretion could be regulated by various v-SNAREs. We also show that the granule cargo decondensation is an intermediate to secretion that also contributes to rates of cargo release. Platelets contain four major VAMP isoforms (-2, -3, -7, and -8), however, VAMP-8 and -7 play a primary role while VAMP-2 and -3 are ancillary in secretion. To exploit this heterogeneity in VAMP usage, platelet-specific V-2/3-/- and V-2/3/8-/- mouse models were generated and characterized to understand how secretion influences hemostasis. We found that each VAMP isoform differentially contributes by altering the rates and extents of cargo release. The loss of VAMP-2 and -3 had a minimal impact while the loss of VAMP-2, -3 and -8 significantly reduced the granule secretion. Platelet activation and aggregation were not affected though the spreading was reduced in V-2/3/8-/- platelets indicating the importance of secretion in spreading. Though coagulation pathways were unaltered, PS exposure was reduced in both V-2/3-/- and V-2/3/8-/- platelets suggesting diminished procoagulant activity. In vivo experiments showed that V-2/3/8-/- animals bled profusely upon tail transaction and failed to form occlusive thrombus upon arterial injury while V-2/3-/- animals did not display any hemostatic deficiency. These data suggest that about 40-50% reduction in secretion provides protection against thrombosis without compromising hemostasis and beyond 50% secretion deficiency, the animals fail to form functional thrombi and exhibit severe bleeding. Additionally, detailed structural analysis of activated platelets suggests that the post-stimulation cargo dissolution depends on an agonist concentration and stimulation duration. This process is VAMP-dependent and represents intermediate steps leading to a full exodus of cargo. Moreover, we also show that VAMP-8 is important for compound fusion events and regulates fusion pore size. This is a first comprehensive report that shows how manipulation of the exocytic machinery have an impact on secretion and ultimately on hemostasis. These animals will be instrumental in future investigations of platelet secretion in many other vascular processes.
3

Genetic predisposition to DTT-induced DNA decondensation

Fouche, Anna Aletta 10 May 2007 (has links)
Male infertility may be due to oligozoospermia, asthenozoospermia and teratozoospermia. Intracytoplasmic sperm injection is used to address male infertility. However, the percentage of viable embryos obtained by this technique is very low. Pronucleus formation has been identified as one of the key events in fertilisation and gamete decondensation is vital for this process to take place. Decondensation can be initiated by chemicals such as DTT that reduce the disulphide groups between the protamine proteins that keep the DNA of the gamete condensed. An increase in decondensation should translate into a higher fertilization rate and a higher yield of embryos. The research from this thesis has compared the decondensation ability via DTT in human spermatozoa and bovine spermatozoa, to study pronucleus formation in bovine zygotes and bovine embryo formation in the presence of DTT; and lastly the cytotoxic effect of DTT using somatic cells in culture has been investigated. In this study 12 semen samples for either fertile or subfertile subjects were collected, isolated and exposed to 25 mM DTT for 0, 5, 7, and 10 minutes, washed and the morphological changes associated with decondensation was evaluated by phase contrast microscopy. After 5 and 7 minutes 11 of the 12 samples underwent decondensation while after 10 minutes several samples showed a lower rate of decondensation and this was associated with and unusual hypercondensed state, CMA3 staining revealed all spermatozoa samples evaluated were mature. However, after treatment with DTT for 5, 7 and 10 minutes an increase in fluorescence was observed indicating increased protamine thiol group reduction and subsequently increased CMA3 accessibility. For some samples reduced fluorescence was observed possible due to the supercoiling of the DNA. DTT successfully induces decondensation of human spermatozoa, however does this lead to the formation of viable embryos? Due to ethical issues associated with working with human embryos all further studies were done using bovine embryos. Spermatozoa used were derived from Friesian bulls and the samples were pooled to prevent sample bias and interindividual variation. Spermatozoa were exposed to 25 mM of DTT at 5, 7, and 10 minutes as used for human spermatozoa. No decondensation was observed using the same conditions as for human spermatozoa, therefore the ‘swim up’ medium containing heparin and regularly used in IVF procedures for bovines was used, and this resulted in successful decondensation of bovine spermatozoa after 30 minutes. The effects of DTT on pronucleus formation and embryo development were evaluated in three bovine specimens. In the first group, DTT had no significant effect on the parameters measured, namely the number of oocytes that were in metaphase II, with one pronucleus, with two pronuclei, with degeneration of the nucleus and polyspermia. In the second group the percentage cleavage and embryo formation was determined on Day 1 (group 2) and 7 (group 3) respectively and statistical differences were obtained between the control and the DTT group. DTT had no significant effect on all the early parameters measured however later in development DTT had a significant adverse effect on cleavage and eventual embryo development. <p)Cleavage and embryo formation is a process of multiple mitotic divisions resulting in an increase in the number of cells that become smaller with each cell division, while somatic cells also undergo mitotic division although the cell size remains constant. Therefore the L929 cell line, a standardized system used to test toxicity, can be used to investigate the toxic effects of DTT on a dividing cell population. In this study L929 cells were expose to 25mM DTT for 30 minutes, and lysosomal membrane integrity, cell viability and number was determined immediately following exposure and after 48 hours growth. In another experiment the L929 cell line was exposed to all concentrations used in this and other studies for 5, 10 and 20 minutes. At all concentrations and exposure times DTT was found to be cytotoxic to the L929 cell line. How exactly DTT mediates this toxic effect is unknown, however due to its high solubility DTT can cross the cell membranes. The tertiary structure of proteins, enzymes and DNA is vulnerable to the reducing effects of DTT. In conclusion, although DTT induces decondensation in human and bovine spermatozoa, in the bovine model it does not lead to viable embryo formation and this has been confirmed in cell culture where DTT at all concentrations used was found to be cytotoxic. / Dissertation (MSc (Anatomy))--University of Pretoria, 2006. / Anatomy / unrestricted
4

Regulace genové exprese ve čtyřech dimenzích / Gene regulation in four dimensions

Vaňková Hausnerová, Viola January 2018 (has links)
Transcription has turned out to be a discontinuous process when imaged at a single cell level. This observation is referred to as transcriptional bursting or pulsing and has been detected in a variety of organisms ranging from bacteria to mammalian cells. The dynamics of transcriptional pulsing are influenced by the properties intrinsic to the transcriptional process, as well as by upstream factors: chromatin environment, signalling molecules, cell cycle stage etc. In the first part of this thesis, we focused on the regulation of transcriptional pulsing in the nucleolus. Using imaging of living cells, we detected pulsatile transcription of a transgene with nucleolar localization whose expression was mediated by RNA polymerase II. In the second part of the thesis, we investigated the relationship between chromatin decondensation and transcriptional dynamics. We used hyperosmotic medium to induce global condensation of chromatin and revealed that upon chromatin decondensation, a transient spike in transcriptional intensity occurs in induvial living cells. Next, we analysed expression of TFRC and POLR2A genes in several cell cycle stages using single molecule RNA FISH. We detected increase in both frequency and size of transcriptional pulses during a limited time window which coincided with chromatin...
5

Multiscale analysis of poly-ADP-ribosylation dependent chromatin remodeling mechanisms at DNA breaks / Analyse multi-échelle des processus de remodelage de la chromatine au niveau des dommages de l'ADN contrôlés par la poly-ADP-ribosylation

Lebeaupin, Théo 18 October 2017 (has links)
Pendant longtemps, la chromatine a été uniquement décrite comme un moyen de compacter près de deux mètres d’ADN dans un noyau de quelques micromètres de diamètre. On sait aujourd’hui que la chromatine représente en fait un élément majeur de régulation de toutes les fonctions nucléaires impliquant l’ADN. Dans le contexte de dommages de l’ADN induits par irradiations UV, la chromatine endommagée subit une décondensation rapide et transitoire qui l’amène à occuper un volume 1,5 fois plus grand que son volume initial. Cette relaxation chromatinienne est associée à une plus grande accessibilité de l’ADN. Néanmoins, le lien entre ces deux effets découlant de la présence de dommages, n’a pas été établi, ni caractérisé. En couplant l’imagerie de cellules vivantes à l’induction de dommages ciblés au sein de noyaux cellulaires par micro-irradiation laser, ces travaux ont permis de mettre en évidence le rôle majeur de PARP1 dans la réponse chromatinienne aux dommages de l’ADN. En effet, certaines conclusions contradictoires présentes dans la littérature scientifique concernant l’action de PARP1 sur la chromatine ont été réconciliées en démontrant que PARP1 seul peut se lier à la chromatine et entraîner une plus forte compaction de celle-ci, tandis que son activité catalytique de PARylation va, quant à elle, conduire à une décompaction de la structure chromatinienne. Cette étude s’est aussi intéressée à la dynamique particulière de l’histone H1 suite aux dommages de l’ADN. En effet, celui-ci est rapidement exclu des zones de dommages par un mécanisme encore inconnu, et les éléments apportés ici suggèrent que H1 pourrait jouer un rôle dans la décondensation de la chromatine suite aux dommages de l’ADN. Pour finir, des techniques de photo-perturbation et de spectroscopie de corrélation de fluorescence ont été employées pour comprendre et caractériser l’environnement moléculaire que constitue la chromatine endommagée et décondensée. Bien qu’une augmentation significative des interactions entre la chromatine et certains de ses partenaires d’interactions soit observée au sein des zones endommagées, aucun changement en termes d’encombrement moléculaire n’a pu être mis en évidence à ce niveau qui pourrait expliquer une plus grande accessibilité de l’ADN. / For a long time, chromatin was only described as a mean to fit the two-meters long DNA molecule into a nucleus of only a few microns. It is admitted today that chromatin actually represents a key element in the regulation of all nuclear functions dependent on DNA. In the context of UV-induced DNA damage, chromatin undergoes a rapid and transient relaxation which leads to an expansion of the damaged area to 1.5 times its original size. While this chromatin response to damage is associated with a higher DNA accessibility, the link between those two phenomena, as well as the mechanisms driving them, are still poorly understood. Using live-cell imaging and laser micro-irradiation to induce DNA damage on specific nuclear areas, this work allowed to gain hindsight on the predominant role played by PARP1 in the DNA damage-induced chromatin relaxation. Indeed, showing that PARP1 at DNA damage sites can both induce chromatin compaction through its recruitment at DNA breaks or chromatin decondensation through its PARylation activity helped reconcile its apparent opposite effects described in the literature. A focus was also made on the linker histone H1, as it displays a peculiar behavior upon DNA damage, being rapidly released from the site of DNA lesions. Even if the driving force behind H1 release from damaged chromatin areas has not been identified yet, its behavior suggests that H1 might play a part in chromatin relaxation or in increasing DNA accessibility upon DNA damage. Lastly, combining photo-activation techniques and fluorescence correlation spectroscopy, experiments were performed in order to understand the physical environment that damaged, relaxed chromatin constitutes. We report here that, while enhanced binding of random DNA binding factors is observed in the damaged chromatin area, no significant change is observed in the macromolecular crowding levels that could potentially explain this enhanced binding, as well as a higher DNA accessibility.
6

Noyau spermatique humatin et fertilité / Human sperm nucleus and fertility

Vorilhon, Solène 05 July 2019 (has links)
Chez l’Homme, les succès de la fécondation et d’un développement embryonnaire aboutissant à la naissance d’un enfant en bonne santé résident principalement dans la qualité des cellules reproductrices. Les dommages oxydants de l’ADN spermatique sont une cause majeure d’infertilité masculine. Afin de permettre une prise en charge thérapeutique optimale et adaptée, j’ai tout d’abord mis au point et validé un test diagnostique de l’oxydation de l’ADN spermatique par immunodétection du 8-hydroxy-2'-desoxyguanosine (8-OHdG), adduit majeur de l'oxydation nucléaire. Ce travail de thèse a déterminé, pour la première fois, un seuil d’oxydation de l’ADN spermatique en relation avec les paramètres conventionnels spermatiques. Dans un second temps, je me suis focalisée sur les atteintes de la chromatine et de l’ADN spermatique les plus fréquentes en cas d’infertilité masculine, à savoir les anomalies de condensation de la chromatine, la fragmentation et l’oxydation de l’ADN spermatique. Une corrélation entre l’oxydation de l’ADN, tout particulièrement la moyenne d’intensité de fluorescence, et le pourcentage de spermatozoïde fragmenté a été mise en évidence. Pour objectiver l’impact de ces dommages nucléaires spermatiques en pratique clinique, j’ai étudié, après cryopréservation, les effets bénéfiques d’une supplémentation en hypotaurine des milieux de sélection et de congélation/décongélation des échantillons. Une baisse de la cryocapacitation et du pourcentage de spermatozoïde fragmenté et décondensé ont été retrouvées ainsi qu’une amélioration de la vitalité et de la mobilité progressive spermatique. Enfin, comme le spermatozoïde a pour but ultime de participer à la genèse d’un nouvel individu, j’ai mis en évidence que la fragmentation et l’oxydation de l’ADN spermatique avaient un impact à des moments clés de la cinétique du développement embryonnaire précoce suite à une ICSI sans pour autant modifier l’obtention de blastocystes de bonne qualité. Ce travail de thèse a permis de mieux comprendre la physiopathologie de l’infertilité masculine et de mettre en évidence de nouveaux biomarqueurs spermatiques en lien avec un développement embryonnaire normal. / In humans, the success of fertilization and embryonic development leading to the birth of ahealthy child lies mainly in the quality of reproductive cells. Oxidative damage to sperm DNAis a major cause of male infertility. In order to provide optimal and appropriate therapeuticmanagement, I first developed and validated a diagnostic test for sperm DNA oxidation byimmunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a major adduct of nuclearoxidation. This thesis work determined, for the first time, a threshold for the oxidation ofsperm DNA in relation to conventional sperm parameters. In a second step, I focused on themost common chromatin and sperm DNA disorders in male infertility, namely chromatincondensation anomalies, sperm DNA fragmentation and oxidation. A correlation betweenDNA oxidation, particularly the mean fluorescence intensity, and the percentage offragmented sperm was found. To objectify the impact of this nuclear sperm damage inclinical practice, I studied, after cryopreservation, the beneficial effects of hypotaurinesupplementation to the selection and freeze/thaw media of seed samples. A decrease incryocapacitation and the percentage of fragmented and decondensed sperm has beenfound, as well as an improvement in sperm vitality and progressive mobility. Finally, sincethe ultimate goal of the sperm cells is to participate in the genesis of a new individual, I haveshown that the fragmentation and oxidation of sperm DNA has an impact at key moments inthe kinetics of early embryonic development following ICSI without modifying the obtainingof good quality blastocysts. This thesis work has led to a better understanding of thepathophysiology of male infertility and the identification of new sperm biomarkers related tonormal embryonic development.

Page generated in 0.1128 seconds