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  • 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

Identification and characterization of a role for the actin cytoskeleton during sporulation in Saccharomyces cerevisiae

Davis, Dana Alan, 1969- January 1998 (has links)
The actin cytoskeleton is essential in yeast and is composed of actin and numerous actin binding proteins. One actin binding protein, encoded by SAC6, is the yeast homolog of human fimbrin, an actin bundling protein (1). Sac6 protein is not essential for viability but is involved in many cytoskeletal functions. One common phenotype cytoskeletal mutants, including the sac6Δ, have is a defect in sporulation. Although this phenotype has been known for some time, the function of the actin cytoskeleton during sporulation is completely unknown. In order to determine the role of Sac6 protein and the actin cytoskeleton for sporulation, I accomplished the following: (1) I identified the terminal arrest point of the sac6Δ during sporulation as being immediately prior to spore wall formation, (2) By analyzing other mutants, I established that a primary function for the cytoskeleton during sporulation is for endocytosis, and (3) I identified an endocytic pathway in vegetative cells having different requirements for the actin cytoskeleton than the classical endocytic pathway. The events occurring during sporulation have been characterized. By using a number of different assays, I determined that the sac6Δ arrests late in the sporulation pathway. Different arrest points were seen depending on strain background used. However, in the SK1 background, a function for Sac6 protein in spore wall formation was identified. By examining other mutations defective for sporulation, I identified sla2Δ and chc1-521 as having sporulation defects similar to the sac6Δ. SLA2 encodes a cytoskeletal protein that has roles in endocytosis and CHC1 encodes the clathrin heavy chain that has roles in membrane trafficking, including endocytosis (2-4). Actin and Sac6 protein are also required for endocytosis (5). These data led to the model that a function of the actin cytoskeleton during sporulation is for endocytosis. An allelic series of actin mutations had previously been analyzed for ability to undergo receptor-mediated endocytosis (6). This data was compared with the sporulation ability of the actin mutations and a strong correlation was identified between these two phenotypes. I determined that endocytosis does occur throughout sporulation and that the sac6Δ has defects in endocytosis during sporulation. In order to better understand the role of endocytosis during sporulation, I analyzed the endocytosis of Ste6 protein. The half-life of this protein is known to be controlled by the endocytic machinery and it is endocytosed constitutively (7). The data obtained from this assay (although not informative with regards to sporulation) suggests that Ste6 protein has different requirements for the actin cytoskeleton than receptor-mediated endocytosis. All endocytosis appears to require the actin cytoskeleton, however this may be the first demonstration that multiple actin-dependent endocytosis pathways may exist in yeast.
2

Regulation of TGFß signaling on tumor cell migration, invasion and stem cell activity in triple negative breast cancer

Dai, Meiou January 2013 (has links)
Basal-like triple negative breast cancers (TNBCs) display poor prognostic features with larger tumor size, higher tumor grade, and an increased risk for lymph node and distant metastasis as well as tumor recurrence. Transforming growth factor beta (TGFβ) is a key regulator of the cellular processes by which breast cancer cells from the primary tumor metastasize to distant organs. However, the molecular mechanisms underlying TGFβ's pro-metastatic effects remain to be fully elucidated. Here, we investigated the role of TGFβ signaling pathway in regulating cell migration, invasion and cancer stem cell self-renewal capacity, which are the initial and critical steps in breast cancer metastasis. Our studies initially identified a novel function for the cell cycle regulator p21 and its binding partner acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-induced TNBC cell migration and invasion in vitro as well as tumor invasiveness in vivo. As p21 can interact with different cyclin and CDK complexes, we investigated whether other cell cycle regulators are also involved in TGFβ-induced tumor progression. We found that TGFβ promotes physical interaction and nuclear co-localization between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins promote tumor growth and locally invasive tumors. In addition, we found that TGFβ can activate cyclin D1/CDK4 complex to promote cancer stem cell activity and self-renewal capacity in TNBC. Together, we have defined p21, cyclin D1 and CDK4 as key downstream regulators of TGFβ tumor-promoting functions. / Le cancer du sein triple-négatif (CSTN) de type basal démontre des signes cliniques caractéristiques d'un mauvais pronostique, tels qu'une taille et un grade plus élevés des tumeurs, un risque augmenté de développer des métastases lymphatiques et distantes, ainsi qu'un plus grand risque de récurrence de la maladie. Le TGFβ est un régulateur clé des procédés cellulaires par lesquels les cellules cancéreuses du cancer du sein se détachent de la tumeur primaire pour former des métastases aux organes distants. Cependant, les mécanismes moléculaires par lesquels le TGFβ accomplit son rôle pro-métastatique restent à être élucidés. Ici, nous investiguons le rôle du TGFβ dans la régulation de la migration cellulaire, l'invasion, et la capacité des cellules souches à se renouveler, qui sont les étapes initiales et critiques à la formation de métastases dans le cancer du sein. Notre étude a tout d'abord identifié une nouvelle fonction pour le régulateur de cycle cellulaire p21 et son partenaire associé, l'acetyltransférase pCAF, comme étant des régulateurs de transcription dont la présence est critique pour la migration et l'invasion cellulaire in vitro et l'invasion tumorale in vivo médiées par le TGFβ pour le CSNT. Le p21 pouvant interagir avec différents complexes de cycline et CDK, nous avons investigué si d'autres régulateurs du cycle cellulaire sont aussi impliqués dans la progression tumorale médiée par le TGFβ. Nous avons trouvé que le TGFβ promeut l'interaction physique et la co-localisation nucléaire entre la cycline D21 et p21. La co-expression de la cycline D1 et de p21 promeut la croissance tumorale et l'invasion locale des tumeurs. De plus, nous avons trouvé que le TGFβ peut activer le complexe cycline D1/CDK4 pour promouvoir l'activité des cellules souches cancéreuses, ainsi que leur capacité de renouvèlement dans le CSNT. Ces résultats nous ont permis de définir p21, la cycline D1, et CDK4, comme des régulateurs clés en aval du TGFβ dans ses fonctions pro-métastatiques
3

The dynamics of cadherin-mediated cell-cell adhesion tissue separation in Xenopus Laevis

Touret, Anne-Sophie January 2013 (has links)
The formation and maintenance of boundaries between embryonic regions are crucial to preserve the organization of the embryo during development. One important parameter that needs to be regulated at the interface between cells of two different tissues is cell-cell adhesion. In the present study, we attempted to understand and characterize the dynamics of cadherin-mediated cell-cell adhesion at the ectoderm-mesoderm boundary of the Xenopus gastrula. At this boundary, it has been recently shown that cells undergo cycles of attachments and detachments as the result of ephrin/Eph signaling (Rohani, Canty, Luu, Fagotto, & Winklbauer, 2011). Using three-dimensional live confocal microscopy, we found that the total levels of cadherin at the membrane do not change reproducibly during local repulsive events between ectodermal and mesodermal cells. Cadherin clusters, however, appear during contact establishment and often begin to fade away prior to the start of cell detachments. While the role of acto-myosin contraction in this process is still not fully elucidated, we found that cortical actin was upregulated during cell detachments, and that phospho-myosin was enriched at the endogenous ectoderm-mesoderm boundary. Furthermore, our data suggest that there may be two pools of activated myosin, and that the one that is most prominent at the boundary is the one involved in global cortical tension. / La formation et le maintien de frontières entre les différentes régions embryonnaires jouent un role primordial pour préserver l'organisation de l'embryon au cours du développement. L'adhérence intercellulaire est l'un des paramètres qui doivent être régulés aux interfaces entre les cellules de différents tissus. Dans la présente étude, nous avons tenté de comprendre et de caractériser la dynamique des cadhérines à la frontière entre l'ectoderme et le mésoderme dans la gastrula de Xénope. A cette frontière, il a été démontré que les cellules effectuent des cycles d'attachements puis de détachements, dictés par la voie de signalisation éphrine/Eph (Rohani et al., 2011). Par le biais de microscopie confocale tridimensionnelle, nous avons constaté que la quantité de cadhérines à la membrane ne variait pas au cours des instances de répulsions locales entre les cellules de l'ectoderme et du mésoderme. Les "clusters" de cadhérines, en revanche, apparaissent dès l'initiation du contact intercellulaire et semblent diminuer d'intensité progressivement, avant que les cellules ne se détachent complètement. Bien que le rôle de la contraction d'actine-myosine ne soit pas encore totalement élucidé, nous montrons que l'actine corticale augmente pendant et après les détachements cellulaires, et que la phospho-myosine est régulée à la hausse à la frontière ectoderme-mésoderme endogène. De plus, nos données suggèrent qu'il pourrait y avoir deux "pools" de myosine, et que celle qui se démarque à la frontière est celle qui est responsable de la tension corticale globale des cellules.
4

Effect of nitric oxide overexpressing endothelial progenitor cells on coronary artery smooth muscle cells

Guber, Sergio January 2013 (has links)
Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), alone or in combination with estrogen treatment, would potentiate the beneficial effects of EPCs in the context of restenosis.We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (hCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of hCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with hCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced Bcl-2/Bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in hCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, hCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs. These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate hCASMC migration through paracrine effects. / La resténose artérielle, qui se produit dans presque 20% des patients ayant subi une angioplastie, est caractérisée par une prolifération excessive des cellules musculaires lisses vasculaires résultant de la disparition du revêtement des cellules endothéliales. Les progéniteurs endothéliaux circulants (EPC) ont la capacité de recoloniser et réparer l'endothélium vasculaire endommagé, ce qui réduit la resténose. L'oxyde nitrique (NO) contribue à la mobilisation et l'activité fonctionnelle des EPCs, et l'oestrogène a été montré pour augmenter le taux circulant des EPCs et d'accélérer le processus de réendothélialisation par les EPCs chez la souris. En outre, le NO est important pour la transduction de la signalisation oestrogène-dépendante et la réendothélialisation. Nous avons proposé que la surexpression d'oxyde nitrique synthase endothéliale (eNOS), seule ou en combinaison avec un traitement à l'oestrogène, potentialiserait les effets bénéfiques des EPCs dans le cadre de la resténose.Nous avons constaté que les EPC humains natifs (hEPCs) n'ont pas d'effet sur la prolifération et la migration in vitro des cellules musculaires lisses de l'artère coronaire humaine (hCASMC), évaluées par l'incorporation de BrdU et le l'essai « wound-scratch » respectivement. En revanche, le donneur de NO SNAP a diminué de façon significative la prolifération et la migration des hCASMCs. Par la suite, des hEPCs ont été soit transfectés avec un plasmide eNOS humain ou stimulés par du 17β-estradiol (E2) avant d'être co-cultivés avec des hCASMCs. Les niveaux d'eNOS totale et phosphorylée évalués par western blot ont été augmentés de 3 - à 3,5 fois dans les hEPCs transfectés avec eNOS ou stimulés avec E2. Cela a été associé à une augmentation de 3 fois de la production de NO, mesuré par immunofluorescence avec DAF-FM diacétate (p <0,05). Des hEPCs surexprimant eNOS ont montré une augmentation du rapport Bcl-2/Bax et le marquage d'annexine V/iodure de propidium a été réduit indiquant une augmentation de la survie. Fait intéressant, nous avons observé une diminution significative (p <0,05) de la migration des hCASMC de 23% pendant la co-culture avec des hEPCs surexprimant eNOS, et de 56% avec des hEPCs stimulés avec E2. Cependant, la prolifération des hCASMC n'a été affectée ni par des hEPCs surexprimant eNOS, ni par les hEPCs stimulés avec E2. Ces résultats suggèrent que la surexpression de eNOS dans des EPCs humains augmente leur survie et améliore leur capacité à moduler la migration des hCASMC via des effets paracrines.
5

Effects of the HIV-1 protein Nef on the stromal cells of mouse peripheral lymph nodes and on mouse keratinocytes

Meunier, Clémence January 2013 (has links)
The HIV-1 protein Nef plays an important role in HIV-1 pathogenesis, both in humans and mouse models. Indeed, transgenic (Tg) mice expressing Nef under the control of the human CD4 promoter develop many phenotypes that closely resemble those of AIDS patients. Using these Tg mice, we have studied the effects of Nef on lymph node stroma, namely on fibroblastic reticular cells (FRCs), and blood and lymphatic endothelial cells (BECs and LECs, respectively). In human patients, a severe and irreversible loss of LN structure and function is observed, and this is correlated with the depletion of FRCs. We show that, in our model, Nef does not significantly change the size of the FRC population, nor does it affect its functions in resting lymph nodes (LNs). We hypothesized that FRCs are supported by lymphoid tissue inducer cells, which prevent their depletion. No fibrosis or loss of structure could be observed in the LNs of Tg mice, contrary to what is seen in human AIDS patients. Nef did, however, cause a localized expansion of BECs and LECs in medullary blood vessels and the subcapsular sinus, respectively, sometimes to the point of completely obstructing these structures. The mechanism driving this expansion is still under investigation. We also studied an unexpected effect of Nef expression on skin keratinocytes. This expression led to the development of an atopic dermatitis-like disease. Atopic dermatitis is one of the most prevalent skin diseases associated with AIDS but, to our knowledge, no HIV-1 protein had previously been directly linked to it. We show here that Nef causes this atopic dermatitis-like disease by inhibiting the Notch1 signalling pathway in keratinocytes. Thus, our data adds to the list of known Nef effects, and provides potential new insights for therapy. / La protéine Nef du VIH-1 joue un rôle important dans la pathogenèse de ce virus, chez les humains et chez des modèles murins. En effet, des souris transgéniques (Tg) exprimant Nef sous le contrôle du promoteur CD4 humain développent des phénotypes très similaires à ceux retrouvés chez les patients atteints du SIDA. Nous avons étudié, chez ce modèle murin, les effets de Nef sur le stroma des ganglions lymphatiques (GL), c'est-à-dire sur les cellules réticulaires fibroblastiques (CRFs) et les cellules endothéliales vasculaires et lymphatiques (respectivement CEVs et CELs). Chez les humains infectés par le VIH-1, une perte sévère et irréversible de la structure et de la fonction des GLs est observée et est corrélée avec la déplétion des CRFs. Nous montrons ici que, dans notre modèle, Nef ne change pas significativement la taille de la population de CRFs et n'affecte pas ses fonctions. Nous proposons que la population de CRFs est maintenue par les cellules inductrices de tissu lymphoïde. Aucune fibrose ou perte de structure n'a été observée dans les GLs des souris Tg, contrairement à ce qui se retrouve chez les patients humains. Par contre, Nef cause une expansion localisée des CEVs et des CELs dans les vaisseaux sanguins médullaires et dans le sinus subcapsulaire, respectivement, parfois au point de complètement obstruer ces structures. Le mécanisme de cette expansion est à l'étude. Nous avons également étudié des effets inattendus de l'expression de Nef dans les kératinocytes de la peau. Cette expression a provoqué le développement d'une maladie similaire à la dermatite atopique. La dermatite atopique est l'une des maladies de peau les plus fréquentes chez les patients atteints du SIDA. Cependant, à notre connaissance, aucune protéine du VIH-1 n'y avait été directement associée à ce jour. Nous montrons ici que Nef cause cette maladie en inhibant la voie de signalisation de Notch1 dans les kératinocytes. Ainsi, nos résultats identifient de nouveaux effets de Nef et fournissent de nouvelles perspectives potentielles pour des thérapies.
6

The distribution of snRNPs in germ cells during the cycle of the seminiferous epithelium of the adult rat /

Moussa, Fuad January 1993 (has links)
Light microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y$ sb{12}$ localized U1, U2, U4/U6 and U5 (spliceosome) snRNPs predominantly to nuclei of germ cells up to step 10 spermatids. The absence of reactivity after step 11 is concomitant with nuclear condensation and transcriptional arrest (Monesi, 1964). Nuclear and cytoplasmic reactivity in pachytene spermatocytes reached a peak at stage XII, coincident with maximal rates of RNA synthesis (Monesi, 1964). Quantitative EM immunogold labeling of Lowicryl embedded testicular sections confirmed the light microscope observations. Immunogold speckles were observed along nuclear chromatin. The chromatoid bodies of spermatids and spermatocytes and the intermitochondrial material of spermatocytes were found to be additional sites of snRNP localization. This co-localization suggests that these dense cytoplasmic structures are related. Anti-U1 snRNP antibodies applied to frozen sections co-localized with spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is usually spliced.
7

Cell-free reconstitution of an endoplasmic reticulum-mediated activation of MAPKSAPK

Rousselle, Etienne January 2002 (has links)
In response to stresses such as the accumulation of misfolded proteins, the Endoplasmic Reticulum (ER) triggers cytosolic and nuclear signalling events, which are integrated by the cell to result in adaptation or apoptosis. The activation of stress-responsive kinases (SAPKs) and the mitogen-activated kinases (MAPKs) are known to have a key influence cell fate determination. We developed a technique to study the regulation of these kinases by the ER in response to drugs inducing protein misfolding. We isolated ER like compartments with a relatively high degree of purity using magnetic beads coated with anti-calnexin antibodies and vie reconstituted in a cell free system the ER signalling induced by different stresses. We demonstrate here that ER-like compartments isolated from cells treated with azetidine-2 carboxylic acid or Tunicamycin differentially activates ERK-1, JNK-1 and p38 in vitro. Moreover, we show that the molecular adaptors Shc and Nck are involved in the ER-mediated regulation of these kinases.
8

Role of iPLA₂ in complement (C5b-9) mediated GEC injury

Cohen, Daniel, 1980- January 2006 (has links)
There are a number of isoforms in the PLA2 superfamily, including secretory PLA2s (sPLA2) cytosolic PLA2s (cPLA 2), and calcium independent PLAS (iPLA2beta-short, beta-long, and gamma). In membranous nephropathy, glomerular epithelial cell (GEC) injury by complement C5b-9 leads to morphological changes in GEC and proteinuria, in association with cPLA2alpha activation. The present study addresses the role of iPLA2 in GEC injury. Complement-mediated release of [3H] arachidonic acid was most significantly augmented in GEC overexpressing iPLA2gamma (GEC-iPLA2gamma) as compared with GEC-Neo control. The accelerated AA release was inhibited by the iPLA2-directed catalytic inhibitor bromoenol lactone (BEL). For comparison, GEC-iPLA2gamma also amplified [3H]AA release after incubation of GEC with H2O2, or chemical anoxia followed by re-exposure to glucose, whereas stable clones overexpressing iPLA2gamma only amplified [3H]AA release after incubation with H2O2. Complement-mediated cytotoxicity (measured by release of lactate dehydrogenase) was attenuated significantly in GEC-iPLA2gamma, as compared with GEC-Neo, and the cytoprotective effect of iPLA2gamma was reversed by BEL and partially by Indomethacin. In keeping with previous results, incubation of GEC-cPLA2alpha with complement increased free [3H]AA. This increase in [ 3H]AA was blocked by BEL, although BEL did not block cPLA2alpha activity in cell extracts in vitro. Thus, in addition to cPLA2alpha, iPLA2gamma may be involved in complement-mediated release of AA. Moreover, activation of cPLA2alpha by complement appears to be, at least in part, dependent on iPLA2gamma. Modulation of iPLA2gamma activity may provide a new approach to reducing GEC injury.
9

The defects in the oocytes of B6.Ytir sex-reversal female mice /

Chen, Hai Ying January 2003 (has links)
B6.YTIR sex-reversed mice with bilateral ovaries develop an apparently normal female phenotype, but their eggs fail to develop after fertilization. Our previous studies have shown that the primary cause of infertility in the XY female can be attributed to the incompetence of its oocytes for postfertilization development rather than their surrounding somatic cells. The objective of this study was to examine the segregation of chromosomes during two successive meiotic divisions in the oocytes from XY females and the oocytes from XX females as a control. Oocyte-cumulus complexes were collected from gonadotropin (PMSG)-primed ovaries and matured in culture. MII-oocytes after culture for 19--21 hours were activated by 5mM SrCl2. The number and arrangement of chromosomes were observed under a light microscope with phase contrast or processed for FISH to identify the X and Y chromosomes under a fluorescence microscope. The oocytes at varying times of culture and after activation were fixed and immunocytochemically stained for observation of microtubule assembly and cell cycle progression.
10

Identification of proteins interacting with the N-termini of USP2 deubiquitinating enzyme isoforms

Lee, Kyung-Joo, 1977- January 2004 (has links)
The ubiquitin system is a major cytosolic and nuclear pathway of proteolysis in all eukaryotic cells. In this pathway, ubiquitin, a 76 amino acid peptide, is covalently ligated to protein substrates by a series of enzymes; E1, E2 and E3. The ubiquitin-attached proteins are commonly targeted for destruction by the 26S proteasome but the ubiquitination may also direct other cellular functions such as endocytosis, leading to proteolysis in the lysosome. Interestingly, a large family of genes encoding deubiquitinating enzymes (UBP; ubiquitin specific processing protease and UCH; ubiquitin C-terminal hydrolase) has recently been identified from different organisms. These enzymes are found to remove ubiquitin from covalent attachments to itself or other proteins in order to regenerate free ubiquitin or to counteract the effects of ubiquitinating enzymes by removing the polyubiquitin chain from the conjugated protein substrate. Dr. Wing's laboratory has recently identified USP2a and USP2b, two germ cell specific UBP isoforms that share a common core region but divergent N-termini. These termini target the USP2b and USP2a to different subcellular locations and also influence substrate specificity. / To identify substrates or interacting proteins that may serve to localize these enzymes, a bacterial two-hybrid assay was performed using a mouse testis cDNA library and the N-termini of the enzymes as baits. Kpna6, an isoform of Karyopherin alpha (also known as importin alpha) and SKD3, the suppressor of K + transport defect protein were found to be possible interacting proteins of USP2a and USP2b respectively.

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