Dissertations / Theses on the topic 'Internalisation'
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1
Hopkins, Gemma V. "The mechanism of desmosome internalisation." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493936.
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Desmosomes are intercellular adhesive junctions providing architectural integrity to tissues that are subject to mechanical stress. During wound healing downregulation of desmosomal adhesion is necessary to facilitate tissue remodeling. Ultrastructural observations suggest that downregulation may occur by internalisation of whole desmosomes but the mechanism of downregulation is not understood. Protein kinase C alpha (PKCα) has been shown to colocalise with desmosomes at the wound edge and to modulate their adhesion. This thesis investigates the internalisation of desmosomes with particular reference to the roles of the cytoskeleton and PKC.
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Fothergill, Thomas. "Receptor signalling and internalisation of papillomaviruses /." [St. Lucia, Qld.], 2006. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe19808.pdf.
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Baptist, Myma Cynthia. "Studies of μ-opioid receptor internalisation." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520291.
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ELISAs were used to investigate the effects of a range of opioid ligands on the internalisation of the T7-tagged μ-opioid receptor (MOR) expressed in HEK 293 cells. The different opioid ligands were seen to vary in their abilities to induce MOR internalisation. The effect of the MOR agonists, DAMGO and morphine on MOR phosphorylation mutants (MOR-T180A and MOR-S363A) expressed in HEK 293 cells was also examined by ELISA. The ability of DAMGO to induce internalisation was significantly inhibited in the MOR-T180A in comparison to the wild type MOR. An N-terminal superecliptic pHluorin-tagged μ-opioid receptor (pHMOR) was generated to study MOR trafficking. Superecliptic pHluorin is a pH-sensitive variant of GFP which emits fluorescence at physiological extracellular pH (7.4) but not in acidic environments such as that occurring in intracellular organelles. In HEK 293 cells expressing pHMOR, fluorescence from pHluorin was only observed at the plasma membrane and this was abolished by lowering the extracellular pH to 6. Raising the intracellular pH with NH4Cl (50 mM) revealed fluorescence in intracellular compartments. The pHMOR construct was shown to be functional as indicated by the robust activation of GIRK channels as well as the rapid decrease in the level of cell surface pHMOR fluorescence following treatment with DAMGO. Total internal reflection fluorescence (TIRF) microscopy demonstrated that the surface expressed pHMORs were very dynamic and displayed characteristics which were not detected by confocal microscopy experiments such as receptor clustering following treatment with DAMGO or morphine. The agonist-activated receptors were also seen to colocalise with DsRed-tagged clathrin. The TIRF microscopy technique together with the pHluorin tagged MOR presents a very useful and promising approach to investigate dynamic changes in cell surface MOR expression in live cells.
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Higgins, Stephen. "Army adventurous training and the internalisation of core values : how leadership behaviours affect the internalisation of motivational regulations." Thesis, Bangor University, 2012. https://research.bangor.ac.uk/portal/en/theses/army-adventurous-training-and-the-internalisation-of-core-values-how-leadership-behaviours-affect-the-internalisation-of-motivational-regulations(fe2c0b2d-c0a8-4c4e-90a9-196551a47df6).html.
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Adventurous Training (AT) within Army Phase One organisations is used to assist in the development of British Army recruit core values . This study measured the internalisation of British Army recruit core values during the AT week at two separate Phase One training organisations. A pre-test, post-test design was used to evaluate recruit (n = 302) motivational internalisation of core values during a structured 5-day training week, where recruits undertook a mixture of rock climbing, caving, canoeing, kayaking, and hill walking activities, and were required to complete tasks in unfamiliar and challenging environmental conditions. Reflecting the influence of the training, Bonferroni corrected, pair-samples, ttests conducted on the Relative Autonomy Index were significant for the motivational internalisation of All core values and four of the six independent core values (Selfless Commitment, Courage, Loyalty and Respect for Others). Further examination at external, introjected and integrated regulations additionally revealed significant results for all core values with the AT week appearing to have the most robust effect on introjected regulation. A second hypothesis was concerned with the effects of the leadership of AT instructors in developing recruit core values and asked specifically whether high levels of transformational leadership behaviours were associated with an enhanced internalisation of core values. Fifty nine instructors took part in the study and four transformational leadership behaviours were hypothesised to be associated with greater gains in the internalisation of all core values. Analyses revealed mixed results regarding individual transformational leadership behaviours; however, individual consideration was found to be the most significant behaviour. The implications for training developments are discussed.
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Battye, Katherine. "Novel photosensitors for use in photochemical internalisation." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.530844.
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Russell, Hugh Hayden. "Molecular basis of epithelial internalisation of Streptococcus pyogenes." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423540.
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Frawley, Lisa Antoinette. "Internalisation of somatostatin agonists and somatostatin{sub2} receptor." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620184.
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Baratti-Elbaz, Catherine. "Internalisation et recyclage du récepteur de la TSH." Paris 11, 2000. http://www.theses.fr/2000PA11T058.
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Le récepteur TSH est un récepteur couplé aux protéines G présentant des spécificités par rapport aux récepteurs homologues de la lutropine (LH) et folliculostimuline (FSH): (i) son volumineux domaine extracellulaire clivé en deux sous-unités (ii) son activité constitutive (iii) l'existence d'auto-anticorps stimulants dirigés contre le récepteur et impliqués dans l'hyperthyroïdisme. Le trafic intracellulaire du TSHR n'était pas documenté. Des lignées permanentes de cellules L exprimant ce récepteur ainsi que des anticorps monoclonaux dirigés contre le récepteur permettent d'étudier sa distribution et son endocytose en microscopie confocale et électronique. Le TSHR est initialement présent sur la membrane plasmique au sens strict et les puits recouverts de clathrine, au niveau desquels il est internalisé. L'internalisation constitutive représente 10% des récepteurs présents à la surface. L'hormone n'augmente que d'un facteur 3 cette endocytose. La majorité du récepteur internalisé est recyclé via des vésicules lisses alors que l'hormone est dégradée dans les lysosomes. Le recyclage est inhibé par la monensine et emprunte une voie indépendante de la cavéoline-1. Les études de microscopie conduites sur les thyrocytes humains en culture primaire montrent une polarisation baso-latérale du récepteur et une voie d'endocytose similaire. Le récepteur LH est lui internalisé massivement puis dégradé dans les lysosomes. Les essais de colocalisation avec le récepteur transferrine démontrent que les récepteurs homologues LH et TSH suivent un trafic intracellulaire différent dans le même système d'expression cellulaire. L'utilisation de récepteurs chimères des TSHR et LHR prouvent que les domaines transmembranaires et intracellulaires contiennent les informations responsables de l'orientation vers la voie de recyclage versus celle de dégradation. Le domaine extracellulaire semble conditionner le taux d'internalisation. La détermination des séquences impliquées est en coursThe TSH receptor is a G protein coupled receptor, with specific characteristics from the two high homologous lutropin (LH) and folliculostimulin (FSH) receptors (i) its large extracellular domain which is cleaved in two subunits (ii) its constitutive activity towards the cAMP transduction pathway and (iii) the existence of stimulating anti-receptor autoantibody implicated in hyperthyroïdism. Seant information is available on the intracellular trafficking of this receptor. Stahly transfected L cells expressing TSH receptor and anti-receptor antibodies were used to study by confocal and electron microscopies its cellular distribution and endocytosis. The TSH receptor was initially localized on the plasmalemma proper and in clathrin-coated pits. Lt was internalized through clathrin-coated vesicles. Constitutive endocytosis represented 10% of cell surface receptor molecules. Endocytosis was increased only 3 fold by the hormone. The majority of internalized receptor recycled to cell surface via smooth vesicles whereas hormone was degraded in lysosomes. This recycling was inhibited by administration of monensin and occurred via a caveolin-1 independent pathway. Microscopic studies repeated in primary cultures of human thyroid cells showed a baso-lateral distribution and a very similar endocytosis pathway. The LH receptor is endocytosed in high proportion and degraded in lysosomes. Colocalization studies with transferrin receptor confirmed that highly homologous LH and TSH receptors exhibit, when expressed in the same cells, very different cellular trafficking properties. The use of LHITSH receptor chimeras showed that transmembrane and intracellular domains contain information orienting the protein toward recycling or degradative pathways. The extracellular domain seems to play a role in the extent of internalization. These observations should now allow the determination of the molecular signals involved in these processes
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DELMOTTE, CAROLINE. "Etude physicochimique et internalisation cellulaire d'un peptide immunogene." Orléans, 1999. http://www.theses.fr/1999ORLE2032.
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Dans le cadre du developpement de vaccins sous-unites, l'immunogenicite de peptides synthetiques contenant un epitope t cytotoxique (np6) et un epitope t auxiliaire (t) issus de proteines du virus de la rougeole a ete etudiee. Le peptide chimere tt-np6 a pour interet d'induire une reponse t cytotoxique en absence d'adjuvants et de composes hydrophobes. Le mecanisme de reconnaissance d'un antigene par les lymphocytes t ainsi que les nouvelles approches vaccinales capables d'induire une reponse immune cellulaire sont decrits succinctement. Pour stimuler in vivo des lymphocytes t cytotoxiques (ctl), l'epitope t cytotoxique doit etre internalise dans le cytosol. C'est pourquoi nous etudions l'internalisation cellulaire et l'affinite pour la membrane plasmique des peptides tt-np6, t-np6 et tt. Les peptides marques par l'oregon green 514 ont ete localises par microspectrofluorimetrie confocale au niveau de la membrane cellulaire. Le peptide le plus immunogene tt-np6 se differencie des peptides tt et t-np6 par une internalisation plus profonde dans le cytosol et une accumulation de type vesiculaire. L'internalisation est directement reliee a la presence du peptide a la membrane plasmique. Des etudes physicochimiques ont ensuite montre que le peptide tt-np6 a une plus grande affinite pour les lipides, qu'il est plus helicogene et plus stable a la proteolyse que t-np6 et tt. L'etude approfondie de la structure de tt-np6 a mis en evidence une propension a s'autoassocier, ce qui favoriserait la resistance a la proteolyse, et elle a permis d'identifier une arginine qui serait responsable d'une perte d'helicite et dont la mutation ameliorerait l'activite biologique du peptide. Enfin, le peptide tt est etudie du point de vue de son affinite pour des alleles humains et de son helicogenicite afin de permettre son application a d'autres epitopes. Ce travail souligne l'importance des analyses physicochimiques dans l'elaboration de vaccins sous-unites capables de stimuler des ctls.
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Braksator, Ellen. "A Study on μ-opioid receptor desensitisation and internalisation." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520290.
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Parton, Robert Glenn. "The binding and internalisation of tetanus toxin by neuronal tissue." Thesis, University of Leicester, 1987. http://hdl.handle.net/2381/35122.
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Stanton, Ian Geoffrey. "Molecular mechanisms of agonist independent internalisation of the P2Y12 purinoreceptor." Thesis, University of Bristol, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.574262.
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The management of G protein-coupled receptor trafficking is essential for the normal function of these receptors. The P2Y12 purinoceptor plays an essential role in the platelet aggregation response, and has been shown to desensitise and rapidly resensitise as a result of agonist stimulation. These processes are tightly regulated, involving receptor kinases, arrest ins and clathrin. Recent studies have suggested that as well as this tightly regulated agonist driven pathway of receptor trafficking there is also an agonist-independent, constitutive pathway. This thesis aims to identify this constitutive pathway, and to discern the mechanisms involved. P2Y12 internalisation was assessed III human platelets by a novel immunofluorescence approach using an anti-P2Y12 antibody. This study found that P2Y12 internalised both in response to ADP but also in the absence of ADP. This observation was also confirmed through the expression of RA-tagged P2Y12 receptor in CRG cells. P2Y12 receptors were found to localise in Rab 4 and Rab 11 positive endosomes, suggesting that the receptor recycles back the membrane post internalisation. This was confirmed through the use of a novel acid strip ELISA protocol. This study found that the mechanisms that regulate the constitutive traffic of P2Y 12 are broadly similar to those that regulate agonist driven traffic. The presence of the C-terminal PDZ binding motif has been shown to be essential for agonist-induced internalisation of P2Y 12, yet constitutive internalisation was unaffected by disruption of this motif. This led to the identification of two amino acid motifs within the C-terrninus that play a significant role in receptor regulation. The importance of these regulatory mechanisms was confirmed through the use of lentiviral P2Y12 constructs in murine bone marrow derived megakaryocytes as a model for platelets. In conclusion, this thesis presents for the first time evidence that the P2Y 12 receptor undergoes constitutive agonist-independent internalisation, and dissects some of the molecular mechanisms that underlie this process. 11
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Bouyarden, Salima. "Intériorisation - Internalisation : les mécanismes de l'émergence d'une identité musulmane européenne." Phd thesis, Université de Strasbourg, 2013. http://tel.archives-ouvertes.fr/tel-01070014.
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Cette thèse portée sur l'émergence d'une identité musulmane européenne aspire à mettre en lumière les mécanismes qui sous tendent ce phénomène social. Au travers de la question de l'identité sociale, nous avons émis l'hypothèse d'une corrélation existante entre les deux concepts d'intériorisation en sciences sociales, et d'internalisation intrinsèque à la mondialisation en économie. La conceptualisation d'un processus d'"intériorisation-internalisation" que nous proposons dans cette thèse, permet d'une part, d'inscrire le processus de construction de cette identité sociale dans le contexte de la mondialisation. D'autre part, elle permet de proposer une nouvelle grille d'analyse quant à la lecture de certains faits sociaux relatifs aux Européens de confession musulmane.
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Herre, Jürgen. "Dectin-1 : receptor internalisation, trafficking and biological effects in macrophages." Thesis, University of Oxford, 2004. http://ora.ox.ac.uk/objects/uuid:bb525976-bfbf-4817-926b-c3686da071b5.
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In host defence, pattern recognition plays an essential role by enabling the immune system to discriminate self from pathogenic non-self. Pattern recognition is mediated by leukocyte expressed pattern recognition molecules (PRMs), which recognise pathogen associated molecular patterns (PAMPs) on pathogens. Phagocytosis is a critical event for anti-microbial defence and its contribution is not limited to the clearance and killing of pathogens, but extends to the activation of adaptive immunity through production of pro-inflammatory mediators and antigen presentation. Anti- fungal immunity is extremely efficient and operates via recognition, phagocytosis and killing of fungal pathogens by leukocytes. We have examined Dectin-1, a non-opsonic pattern recognition receptor that recognises live fungi and fungal derived particles and that is highly expressed on various leukocyte populations. We wanted to establish whether Dectin-1 contributes to anti-fungal defence by analysing various aspects of the receptor biology. Using both confocal microscopy and flow-cytometry, we demonstrate that Dectin-1 is a phagocytic receptor. Furthermore, using cell lines expressing receptor mutants, we show that this capacity is mediated by the membrane proximal tyrosine residue located in the ITAM-like motif. This makes Dectin-1 the first described phagocytic leukocyte expressed receptor for unopsonised fungi and fungal derived particles, and the first pattern recognition receptor that mediates phagocytic uptake through a tyrosine based motif. We demonstrate that the mechanisms by which Dectin-1 mediates cytoskeletal activation and actin polymerisation are novel, and not shared with the canonical IT AM containing Fc(gamma)Rs. In particular the observation that Syk kinase plays not role in Dectin- 1 mediated phagocytosis in macrophages. We show that Dectin-1 mediates cellular activation in response to zymosan particles and that these (beta)-glucan dependent biological effects require collaboration with toll-like receptors (TLRs) at the cell surface. We also show that ligand size determines intracellular receptor trafficking following internalisation. Furthermore, we show that when biologically active soluble glucans are internalised by Dectin-1, the receptor is retained intracellularly yet, when biologically silent glucans are used, Dectin-1 is recycled. Dectin-1 is thus established as both an important phagocytic fungal pattern recognition receptor with pro- inflammatory abilities and an additional tool with which to study the diversity of signalling processes associated with leukocyte expressed receptors.
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Karikari, Thomas K. "Distinct conformations, aggregation and neuronal internalisation of different tau strains." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/99422/.
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A shared property of several neurodegenerative diseases is the neuronal accumulation of aggregated tau protein. These include Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Many studies have suggested that aggregated tau accumulation in AD brains involves: (i) internalisation of extracellular tau (aggregated or not) into neurons; (ii) induction of endogenous tau aggregation by the internalised tau; and (iii) secretion of part or whole of this aggregated tau complex. This complex then initiates a new cycle of internalisation, aggregation and secretion. While this AD mechanism has strong evidential support, it is unclear if it applies to FTD. It was therefore investigated if and how two FTD-associated tau mutations, V337M and N279K, affect in vitro wild type (WT) tau aggregation and conformation, and studied the cell biological effects of their respective extracellular oligomers. A library of 43 plasmids for expressing full-length and truncated tau and their FTD variants were created, in conjunction with the establishment of a new high-yield tau purification method. Consequently, in vitro biochemical assays showed that the FTD variants distinctively altered the immunological reactivity, the stages of aggregation, and the structural phenotypes of aggregated WT tau four-repeat domain, K18. Internalisation of WT and FTD tau K18 extracellular oligomers was significantly different in human neuroblastoma cells and human stem-cell derived neurons. Internalisation seemed to occur by endocytosis, and the internalised oligomers localised to the nucleus and cytoplasm of human neuroblastoma cells and the soma and neurites of stem cell-derived neurons. Moreover, internalised oligomers co-localised with endogenous tau and the nuclear protein nucleolin, without inducing cell death. These findings provide new perspectives to the cell-to-cell propagation theory of aggregated tau, by demonstrating that cellular internalisation of tau variants may be tightly regulated by the given protein’s folding and aggregation characteristics. This may help to explain several enigmatic aspects of the molecular pathogenesis found in different tauopathies.
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Wang, T. W. "Studies of photochemical internalisation : mechanisms and strategies in cancer therapeutics." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19572/.
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Photochemical internalisation (PCI) is a technique which enhances targeted drug delivery using light in combination with a photosensitiser. It is a modification of photodynamic therapy (PDT) which involves sub-lethal photodynamic treatment to modify the intracellular distribution of co-administered drugs and other agents which are sequestered in lyso/endosomes. In this study, PDT and PCI using a sulfonated chlorin, AmphinexTM (TPCS2a), in combination with two anti-cancer drugs, saporin or bleomycin, have been investigated both in vitro and in vivo. In vitro experiments initially examined the cell uptake, photostability and cellular localisation of Amphinex using A431 human epidermoid carcinoma cells and HN5 human head and neck squamous cells. The cell killing effect after PDT and PCI in the presence of saporin and bleomycin were assessed. The bioavailability and therapeutic efficacy were also compared with another two PCI photosensitisers, TPPS2a and AlPcS2a. The results indicate that PCI is able to induce the relocalisation of bleomycin and saporin inside cells and thereby enhance cell death. A new porphyrin-peptide bioconjugate of a cell penetrating peptide (CPP) and tetraphenylporphine was investigated for PDT and PCI. It was found that CPP peptide conjugation renders efficient cellular delivery of the tetraphenylporphine for use as a PDT and PCI sensitiser. The pharmacokinetics of Amphinex in normal and tumour-bearing rats was studied using quantitative fluorescence microscopy and chemical extraction. In vivo Amphinex PCI effects and the comparison with PDT were investigated in normal rat liver, colon and a syngeneic rat fibrosarcoma tumour models. Amphinex PCI showed a significant enhancement in inducing damage to normal tissues and tumour. The involvement of apoptosis was also established using the TUNEL assay. This thesis has demonstrated that Amphinex has favourable properties for PDT and PCI. The consistent results from both in vitro and in vivo experiments indicate that Amphinex has the potential for further clinical utilization.
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Tapia, Moore Ernesto. "Théorie de l'agence et internalisation incrémentale dans les PME managériales." Aix-Marseille 2, 2008. http://www.theses.fr/2008AIX24009.
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L’internationalisation des PME managériales est abordée par un cadre théorique mixte composé de la théorie de l’agence (courant principal /agent avec un focus par les coûts d’engagement) et du modèle Uppsala (processus d’internationalisation). La thèse répond à la question « pourquoi la personne responsable de l’internationalisation dans une PME managériale va-t-elle engager l’entreprise qui l’emploie dans un processus d’internationalisation standard ? ». Les principaux résultats démontrent que l’internationalisation standard est l’internationalisation incrémentale du modèle Uppsala ; que l’agent en charge de l’internationalisation instrumentalise le processus d’internationalisation incrémentale, aidé en cela par le dispositif de soutien à l’internationalisation, dans l’objectif sinon d’établir une rente, du moins se prémunir d’une perte d’emploi ; que la distance psychique et la perception du risque sont corrélées et interagissent de façon sélective. Les données ont été collectées à l’aide d’un questionnaire de type psychométrique, diffusé par média électronique auprès de 6000 adresses, pour une collecte de 165 questionnaires utiles. Ce travail est l’un des premiers à aborder l’internationalisation des PME par ce cadre théorique, à étudier l’engagement comme explication de l’internationalisation, ainsi que l’interaction entre le développement de la connaissance et l’engagementThe internationalization of managerial SMEs is studied from a joint theoretical standpoint composed of agency theory (principal/agent stream focused on bonding costs) and the Uppsala model of internationalization process. The thesis provides an answer to the question “why would the person in charge of the internationalization use standard internationalization procedures for the managerial SME that employs him/her ?”. Our main results show that standard internationalization is in fact the Uppsala model’s incremental internationalization; that the agent in charge of internationalization instrumentalizes incremental internationalization, encouraged to do so by the internationalization-supporting organizations, in a form of rent-seeking and hedging work-loss; that psychic distance and perception of risk are correlated and interact selectively. Data was collected through a psychometric questionnaire on electronic media. The forms were addressed to 6000 people rendering 165 usable ones. This work is one of the first to consider SME’s internationalization through this particular theoretic perspective, as well as to study commitment as an explanation for internationalization and the interaction between knowledge development and commitment
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Subtil, Agathe. "Endocytose des recepteurs de l'interleukine 2 : internalisation et trafic intracellulaire." Paris 6, 1996. http://www.theses.fr/1996PA066702.
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L'endocytose permet l'entree dans les cellules de molecules du milieu extracellulaire et de constituants de la membrane plasmique. Certaines molecules sont internalisees apres fixation a un recepteur specifique, par endocytose par recepteur. Pour la plupart des recepteurs etudies jusqu'a present, ce processus a lieu au niveau de puits recouverts d'un manteau proteique de clathrine. D'autres voies d'internalisation existent mais sont mal documentees. Apres endocytose, les recepteurs peuvent etre recycles vers la membrane plasmique, ou orientes vers d'autres compartiments intracellulaires. Ce tri met en jeu des signaux encore peu connus. Nous avons etudie l'endocytose du recepteur d'une cytokine, le recepteur de l'interleukine 2 (il2), dans les lymphocytes. Le recepteur de haute affinite pour l'il2 est compose de trois chaines, alpha, beta et gamma, qui sont internalisees avec leur ligand. Nous avons montre que dans des conditions ou l'entree du recepteur de la transferrine, un marqueur de la voie dependante de la clathrine, etait inhibe, l'endocytose du recepteur de l'il2 demeurait rapide. Ce resultat suggere que l'il2 peut etre internalisee par un mecanisme independant de la clathrine. Une etude par microscopie confocale nous a permis de montrer qu'apres internalisation, les trois chaines du recepteur sont orientees differemment : la chaine alpha recycle vers la membrane plasmique, tandis que les chaines beta et gamma vont vers des compartiments de degradation. Nous avons etudie les signaux moleculaires responsables de l'orientation de la chaine beta vers la degradation. Ce travail a abouti a la caracterisation d'un motif de dix acides amines suffisant pour orienter une proteine membranaire vers la degradation. Ce motif est structure en helice amphiphile. Il ne ressemble pas aux signaux de tri decrits jusqu'a present.
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Chitray, Melanie. "Investigating potential factors affecting foot-and-mouth disease virus internalisation." Diss., University of Pretoria, 2008. http://hdl.handle.net/2263/30391.
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Foot-and-mouth disease (FMD) is a highly contagious disease caused by the FMD virus (FMDV) belonging to the Picornaviridae family. The virus affects cloven-hoofed animals and occurs as seven immunologically distinct serotypes where six of the seven serotypes occur in Africa. This fact, as well as the role of wildlife in virus maintenance, makes eradication and control of FMDV in Africa difficult. Thus, it is imperative to attain more information regarding the genetic diversity of FMD viruses prevalent on the African continent to further our knowledge of the virus as well as to enable better control strategies and the development of improved vaccines. Sufficient genetic information regarding the Leader (L) and complete capsid-coding, P1, region of serotype A and O viruses prevalent on the African continent is lacking, although the SAT isolates have been extensively characterised in the past. In this study the sequence of the L/P1-coding region was successfully determined using a genome-walking approach for a small number of A and O viruses recovered from outbreaks isolated from various species in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions 1A, 1B, 1C and 1D revealed that the African isolates grouped strictly according to serotype and geographic region which indicated the possibility of transboundary spread of the virus within East and West African countries respectively. In contrast, phylogenetic analysis of the non-structural, Lpro-coding region revealed a different tree topology compared to the capsid-coding regions for the A and O isolates with sub-grouping according to serotype and geographic regions was less apparent. The relatedness between the serotype A and O L region might be the result of genetic recombination. The inter and intratypic nucleotide and amino acid variation of the A and O isolates revealed that the most variable capsid-coding region was the externally located VP1 whilst the internally located VP4 capsid protein was the most conserved. The observed variation is in agreement with other studies and reflects the selective pressures on these proteins which either allow or prevent the occurrence of genetic changes for structural constraints or immune escape. Surprisingly, the L protease-encoding region also displayed a high degree of variation. A detailed analysis of the L/P1 amino acid alignment of the A and O isolates revealed that although the extent of variation is high in these regions, the amino acids identified in previous studies as important for FMDV structure (for the capsid-coding regions) and function were found to be conserved, indicating that the virus has adapted itself to elude the host immune response without affecting its vital functional and structural abilities. Additionally, it was observed that the amino acid residues identified as being important for FMDV attaching to the host cell receptors e.g. the RGD amino acid motif of VP1 was highly conserved for all isolates. To further investigate the FMDV-receptor interaction, RT-PCRs were developed to examine the mRNA expression level of the known FMDV receptors. The â integrins that facilitate FMDV cell entry i.e. â1, â3, â6, â8 and heparan sulphate proteoglycans (HSPG) were investigated in susceptible cell lines used for FMDV vaccine production i.e. IB-RS-2 and BHK-21. The RT-PCRs were successfully developed and optimised. The results showed that the mRNA expression levels were variable for all receptor cDNAs tested across 36 passage levels of IB-RS-2 and BHK-21 cells. No distinct differences in virus susceptibility for three FMDV strains with continuous cell passage of IB-RS-2 and BHK-21 cells at passage levels 5, 21 and 36 could be found. The information gained from this study regarding the viral L and P1 region genetic diversity, and phylogenetic analysis has indeed impacted on our understanding of FMDV African viruses. Additionally, the investigation of the FMDV receptor mRNA expression levels and virus susceptibility on two cell lines with continuous cell passage has proved a vital starting point to determine the possible receptors expressed on the surface of cells used by the vaccine production division at the ARC-OVI-TADP and forms the basis for further investigations of the FMDV receptors on the protein level and the development of a real-time RT-PCR for FMDV receptor expression.Dissertation (Msc)--University of Pretoria, 2008.
Veterinary Tropical Diseases
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Dinnis, Diane. "Agonist-induced internalisation of the vasoactive intestinal polypeptide receptor (VPAC2)." Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/22151.
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To investigate agonist-induced internalisation of the VPAC2 receptor we generated a C-terminally epitope-tagged receptor (VPAC2-HA) and mutant receptors with serial C-terminal truncations. Addition of the epitope tag had no significant effect on the second messenger signalling or agonist binding properties of the VPAC2 receptor stably expressed in the human embryonic kidney cell line (HEK293). Iodinated helodermin, a VPAC2 receptor agonist, was rapidly internalised in cells expressing the VPAC2 and VPAC2-HA receptors following incubation at room temperature or at 37°C. Internalisation kinetics of the VPAC2 and VPAC2-HA receptor were indistinguishable. In serum starved cells, the epitope-tagged VPAC2 receptor was predominantly located in the plasma membrane. Treatment with VIP caused a marked shift in receptor distribution from the plasma membrane to a single intracellular site. Receptor internationalisation was dependent upon the concentration of agonist, incubation time and temperature. Removal of agonist resulted in the reappearance of receptor immunoreactivity at the plasma membrane; this movement was unaffected by the presence of the protein synthesis inhibitor cycloheximide, suggesting that the majority of receptors are recycled. Other work in our laboratory has demonstrated that truncation of the C-terminal intracellular domain of the VPAC2 receptor prevents agonist-induced receptor phosphorylation. Nonetheless, truncated receptors were still able to internalise, indicating that phosphorylation is not an absolute requirement for VPAC2 receptor internalisation. The truncated VPAC2 receptor did not desensitise so we postulate that phosphorylation is necessary for this phenomenon. VPAC2 receptors colocalise with transferrin receptors, which are internalised via clathrin pits and act as a marker of endosomes. No colocalisation was observed between the VPAC2 receptor and a marker for the trans-golgi network, indicating that the receptor does not recycle through this compartment. This work provides the first direct evidence for agonist-induced internalisation of the VPAC2 receptor.
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Kisanga, Neema, and Samana Mohammad. "Entry mode and institutional conditions to consider when entering a new market : The case of fashion apparel franchising in Germany." Thesis, Internationella Handelshögskolan, Högskolan i Jönköping, IHH, Företagsekonomi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:hj:diva-43960.
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Background: Literature suggests that franchising as an entry mode for internalisation gains more and more popularity. However, existing literature shows many studies concerning franchising do not focus on industries. Hence, very little research is done when it comes to franchising in the low-to-medium cost fashion apparel industry. At the same time, the growing fashion apparel industry is becoming more and more important due to the business opportunity it brings for organisations. In this context, Germany as being the biggest apparel market in Europe is attractive for international organisation to expand to. For entering the German market through the franchising entry mode, the information about underlying market environment and relevant actors play a vital role to reduce risk of encountering uncertain obstacles in the process. Purpose: Entering a new market as a franchisor can be challenging due to different dynamics that can be found in different markets. Therefore, the purpose of this thesis is to explore institutional conditions of the current fashion apparel industry in Germany and to find out which institutions in Germany could help an organisation in terms of information on prevailing conditions, to successfully enter the German market. Method: To attain the purpose of the research, a qualitative approach employing a single case with two embedded units of analysis is used. Purposive sampling is used to select research participants based on their expertise about the topic. The empirical data is collected through semi-structured interviews with 12 participants, which resemble 3 different actors, the German consumers, the German Franchise Association and Tijarat AB, a fashion apparel company seeking to expand to Germany. Supplementary data, such as official governmental and associations website, is used to support the empirical findings. Secondary data is acquired using literature, web sources and legal documents. The empirical findings are analysed with the help of the thematic analysis and the institutional theory as well as the Uppsala internalisation model. Findings: The empirical findings present that there are several normative conditions which depict behaviours and what is considered to be acceptable in the German market. Firstly, there is no franchise fee collected by the franchisor in the fashion apparel industry. Furthermore, brand awareness, consistency, reputation and quality as well as price, design and variety play an important role in the consumer shopping behaviour and decisions. It was also found that there is no specific franchise law but rather a combination of existing legislation, such as the German Civil and Commercial Code, Competition law, Consumer law and Unfair trade law that form the jurisdiction for franchising in Germany.
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Beaumont, Vahri. "Desensitisation of somatostatin receptors in NG108-15 cells." Thesis, University of Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246243.
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Desaunay, Aurélien. "Etude et modélisation de la biosorption des métaux par les bactéries. Application au transfert du cadmium et du zinc, seuls ou en mélange par Escherichia coli et Cupriavidus metallidurans en colonnes de sable d'Hostun." Thesis, Grenoble, 2011. http://www.theses.fr/2011GRENU049/document.
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Recent field observations have demonstrated that supposedly poorly mobile metals can be detected at long distances from their source, highlighting the importance of poorly predicted transport processes. The fast mobilisation of metals by the colloidal and mobile fraction of soils and in particular biotic colloids (bacteria, algae, fungi, virus, etc.), is now identified as an important secondary transport process that can lead, under specific conditions, to accelerated and potentially dominant pollutant transfer towards aquifers. In order to better understand the role of the bacterial compartment of soils to metal leaching, we conducted a coupled study under static and dynamic conditions. Firstly we evaluated Zn and Cd metal biosorption onto active or inactive Gram negative bacteria (Escherichia coli and Cupriavidus metallidurans CH34) by characterizing the sub-cellular distribution of the metals through a cell disruption approach. The quantification of Zn and Cd in extracellular, membrane and cytoplasm compartments of the cells permitted to show that metals are unequally distributed between the three cell compartments and also between the two bacteria. Surprisingly, metals internalization appeared to be the dominant accumulation process of metals (high cytoplasm contents). The physiological state of the cells was also shown to be important in metal management by the bacteria, since metal accumulation in active cells was reduced due to enhanced efflux and/or EPS production mechanisms. These results suggest bacteria can internalize important amounts of heavy metals and also that adsorption onto cell surface is only a first step in metal management by bacteria. The so-determined thermo-dynamic reactivity constants were used to fit metal breakthrough curves performed in natural sand columns. The transport experiments of bacterial cells, metals or mixtures of bacteria and/or metals performed in the second part of the study, demonstrated that bacteria are able to accelerate the in situ mobilization of Cd and Zn retained in natural sand columns. This transport process was shown to be dominant upon aqueous transport and was correctly fitted using a combined transfer and geochemical modelling approach. Altogether, these results showed that, under specific conditions, heavy metal transport by bacterial cells can dominate aqueous transport processes in soilsRecent field observations have demonstrated that supposedly poorly mobile metals can be detected at long distances from their source, highlighting the importance of poorly predicted transport processes. The fast mobilisation of metals by the colloidal and mobile fraction of soils and in particular biotic colloids (bacteria, algae, fungi, virus, etc.), is now identified as an important secondary transport process that can lead, under specific conditions, to accelerated and potentially dominant pollutant transfer towards aquifers. In order to better understand the role of the bacterial compartment of soils to metal leaching, we conducted a coupled study under static and dynamic conditions. Firstly we evaluated Zn and Cd metal biosorption onto active or inactive Gram negative bacteria (Escherichia coli and Cupriavidus metallidurans CH34) by characterizing the sub-cellular distribution of the metals through a cell disruption approach. The quantification of Zn and Cd in extracellular, membrane and cytoplasm compartments of the cells permitted to show that metals are unequally distributed between the three cell compartments and also between the two bacteria. Surprisingly, metals internalization appeared to be the dominant accumulation process of metals (high cytoplasm contents). The physiological state of the cells was also shown to be important in metal management by the bacteria, since metal accumulation in active cells was reduced due to enhanced efflux and/or EPS production mechanisms. These results suggest bacteria can internalize important amounts of heavy metals and also that adsorption onto cell surface is only a first step in metal management by bacteria. The so-determined thermo-dynamic reactivity constants were used to fit metal breakthrough curves performed in natural sand columns. The transport experiments of bacterial cells, metals or mixtures of bacteria and/or metals performed in the second part of the study, demonstrated that bacteria are able to accelerate the in situ mobilization of Cd and Zn retained in natural sand columns. This transport process was shown to be dominant upon aqueous transport and was correctly fitted using a combined transfer and geochemical modelling approach. Altogether, these results showed that, under specific conditions, heavy metal transport by bacterial cells can dominate aqueous transport processes in soils
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Wills, Lauren. "SUMOylation of the B2AR influences receptor internalisation, desensitisation and downstream signalling." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8564/.
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The beta 2 adrenergic receptor (β2AR) is a GPCR that is susceptible to multiple post-translational modifications (PTMs) including phosphorylation, ubiquitination, palmitoylation and glycosylation, which can alter how the β2AR orchestrates downstream intracellular signals. Following the discovery of SUMOylation of the cardiac signalling protein SERCA2a, we hypothesised that the β2AR, which is also involved in cardiac signalling, may be a substrate for SUMOylation. This notion was supported by previous findings that five different GPCRs have been identified as susceptible to SUMOylation, including metabotropic glutamate receptors (mGluR), a cannabinoid receptor, a serotonin receptor and a receptor involved in basal cell carcinoma known as smo. For the first time, we confirm the susceptibility of the β2AR to SUMOylation by identifying SUMO accepting lysines, interaction sites between the β2AR and the enzymes of the SUMOylation cascade, and the traditional “ghost” band, which is indicative of protein SUMOylation. SUMOylation has been shown to influence receptor signalling, and we now uncover a possible role for SUMOylation in β2AR signalling. I report that SUMOylation of the β2AR (mediated via overexpression of the E3 ligase PIASγ) reduces β2AR phosphorylation by PKA altering the receptor driven phospho-ERK response, inhibits β2AR ubiquitination and degradation, and delays β2AR internalisation. These changes could be associated with steric effects of the bulky SUMO modification and ubiquitin-SUMOylation competition for available surface associated lysines. With the importance of SUMOylation in multiple disease states (including cardiovascular disease, neurodegenerative disease and cancer), we worked in conjunction with the antibody production company Badrilla ® to produce a SUMO-substrate specific antibody for a site within the third intracellular loop of the β2AR. The antibody we have produced is successful in recognising the SUMOylated form of the receptor in both cell and tissue lysate, making it the first antibody of its kind to be generated. In conjunction with the Hajjar group at Mount Sinai Cardiovascular Research Centre (New York, USA) we used the SUMO-β2AR specific antibody to assess the influence of the SUMO modification in two animal models of heart failure (HF); transverse aortic constriction (TAC) pressure overload HF model in mice and the left anterior descending (LAD) artery balloon occlusion ischemic HF model in pigs. Prior work within the Hajjar group has revealed that SUMOylation of SERCA2a is reduced in HF, and restoring this modification to SERCA2a via SUMO-1 adeno-associated viral-mediated gene delivery is beneficial in restoring cardiac function. We hypothesised that SUMOylation of the β2AR would also be reduced in HF, however unexpectedly; the SUMOylated form of the β2AR was increased in the LAD artery balloon occlusion ischemic HF model in pigs. The role of the β2AR in HF is unclear. There are studies which both report a cardio-protective and a cardio-toxic role of the receptor. To ascertain the role SUMOylation of the β2AR plays, in vivo studies promoting SUMOylation of the β2AR in the HF models described above will help to determine if enhanced SUMOylation of the receptor worsens the HF phenotype or prevents its development. To conclude, we present the first evidence that the β2AR can be modified by SUMOylation, which acts to influence the receptors downstream signalling, desensitisation and degradation. We have designed a first-in-class SUMO-β2AR antibody – which paves the way for a panel of SUMO specific antibodies – and utilised it to assess SUMOylation of the β2AR in model cellular systems and animal models of HF. Similarly, to SERCA2a, SUMOylation does influence β2AR in the HF phenotype, although not a decrease in SUMOylation as was expected, but an increase. Future work in animal models promoting SUMOylation of the receptor is vital to assess the role of this highly novel post-translational modification of the β2AR.
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Patel, Waseema Salim. "The role of intracellular Ca2+ in cigarette smoke-induced CFTR internalisation." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3955.
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Chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide, is characterised by airflow obstruction and is primarily caused by smoking. In contrast, another obstructive pulmonary disease, cystic fibrosis (CF), has orphan disease status. However, patients with either COPD or CF present with similar clinical lung problems. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activity is reduced in both diseases. Recent work from our lab showed that cigarette smoke-induced increases in cytosolic Ca2+ were consequential in reducing plasma membrane expression of CFTR by an unknown mechanism. Therefore, the major aim of my project was to identify the molecular mechanism underlying the loss of CFTR activity brought about by an increase in cytosolic Ca2+. Whole cell patch clamp recordings in HEK 293T cells transiently transfected with CFTR showed that an increase in cytosolic Ca2+ significantly reduced CFTR-mediated conductance. Characterisation of the dynamic changes in cytosolic Ca2+, induced by a range of agonists, showed that a sustained increase in Ca2+ was not essential for the loss of CFTR-mediated conductance, but it did involve a dynamin-dependent internalisation of the channel. Confocal imaging further confirmed that an increase in cytosolic Ca2+ caused a reduction in plasma membrane CFTR expression, and a reciprocal increase in intracellular CFTR. Activation of the MEK/ERK pathway has previously been linked to smoke-induced internalisation of CFTR. Similarly, inhibition of the pathway prevented a Ca2+-induced internalisation of CFTR, indicating this pathway also plays a role in Ca2+-induced CFTR internalisation. Importantly, inhibition of the Ca2+ dependent phosphatase calcineurin with cyclosporin A prevented both Ca2+ as well as smoke-induced loss of CFTR, suggesting that the mechanism of internalisation is linked to dephosphorylation, possibly of CFTR itself. Furthermore, either an increase in Ca2+, or exposure to cigarette smoke, increased calcineurin activity, further implicating this phosphatase as a key effector. Functionally, inhibition of calcineurin prevented against a smoke-induced reduction in ASL height whilst having no effect on physiological changes in height induced by G protein-coupled receptor agonists; signifying calcineurin only gets activated under conditions of stress. These findings highlight a role for cytosolic Ca2+ in modulating CFTR activity. Additionally, these data may lead to novel therapeutic strategies aimed at correcting ASL hydration in smokers as well as in people with CF.
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Giger, Florence. "Internalisation mechanisms of the endoderm during gastrulation in the zebrafish embryo." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066203/document.
Full textAbstract:
Au cours du développement, les cellules sont progressivement séparées dans des territoires distincts délimités par des frontières embryonnaires. La première ségrégation a lieu pendant la gastrulation, quand l’embryon s’organise en trois feuillets embryonnaires, l’ectoderme, le mésoderme et l’endoderme. Les mécanismes moléculaires et cellulaires assurant cette ségrégation n’ont pas encore été élucidés. Au cours de ma thèse, je me suis focalisée sur l’internalisation de l’endoderme chez le poisson-zèbre. À partir de résultats in vitro, il a été suggéré que les progéniteurs de feuillets embryonnaires soient ségrégés par un tri cellulaire passif. En combinant des expériences de transplantation de cellules, une imagerie confocale en temps réel et des analyses fonctionnelles, j’ai montré que l’internalisation des cellules endodermiques est due en réalité à un processus de migration active dépendante de Rac1 et de son effecteur Arp2/3, un régulateur direct de l’actine. De manière surprenante, les cellules endodermiques ne sont pas attirées par leur destination interne, mais semblent plutôt migrer hors de leurs voisines. Ce processus est dépendant de la voie Wnt/PCP et de la N-cadhérine. De plus, la N-cadhérine est suffisante pour induire l’internalisation de cellules ectodermiques, sans modifier leur identité. Dans leur ensemble, ces résultats conduisent à un nouveau modèle de formation des feuillets embryonnaires dans lequel les cellules endodermiques migrent activement hors de l’épiblaste pour atteindre leur position interne dans l’embryonDuring development, cells are progressively separated into distinct territories, delimited by embryonic boundaries. The first segregation event occurs during gastrulation, when the embryo is organised in three germ-layers, the ectoderm, the mesoderm and the endoderm. The molecular and cellular mechanisms ensuring this segregation have not yet been elucidated. During my PhD thesis, I have focused on the endoderm internalisation in the zebrafish embryo. Based on in vitro results, it has been suggested that germ-layer progenitors would be segregated by a passive cell sorting. Combining cell transplantation, live confocal microscopy and functional analyses, I have shown that endodermal cell internalisation actually results from an active migration process dependent on Rac1 and its effector Arp2/3, a direct regulator of actin. Strikingly, endodermal cells are not attracted to their internal destination but rather appear to migrate out of their neighbouring cells. This process is dependent on the Wnt/PCP pathway and N-cadherin. Furthermore, N-cadherin is sufficient to trigger the internalisation of ectodermal cells, without affecting their fate. Overall, these results lead to a new model of germ-layer formation, in which endodermal cells actively migrate out of the epiblast to reach their internal position
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Giger, Florence. "Internalisation mechanisms of the endoderm during gastrulation in the zebrafish embryo." Electronic Thesis or Diss., Paris 6, 2016. http://www.theses.fr/2016PA066203.
Full textAbstract:
Au cours du développement, les cellules sont progressivement séparées dans des territoires distincts délimités par des frontières embryonnaires. La première ségrégation a lieu pendant la gastrulation, quand l’embryon s’organise en trois feuillets embryonnaires, l’ectoderme, le mésoderme et l’endoderme. Les mécanismes moléculaires et cellulaires assurant cette ségrégation n’ont pas encore été élucidés. Au cours de ma thèse, je me suis focalisée sur l’internalisation de l’endoderme chez le poisson-zèbre. À partir de résultats in vitro, il a été suggéré que les progéniteurs de feuillets embryonnaires soient ségrégés par un tri cellulaire passif. En combinant des expériences de transplantation de cellules, une imagerie confocale en temps réel et des analyses fonctionnelles, j’ai montré que l’internalisation des cellules endodermiques est due en réalité à un processus de migration active dépendante de Rac1 et de son effecteur Arp2/3, un régulateur direct de l’actine. De manière surprenante, les cellules endodermiques ne sont pas attirées par leur destination interne, mais semblent plutôt migrer hors de leurs voisines. Ce processus est dépendant de la voie Wnt/PCP et de la N-cadhérine. De plus, la N-cadhérine est suffisante pour induire l’internalisation de cellules ectodermiques, sans modifier leur identité. Dans leur ensemble, ces résultats conduisent à un nouveau modèle de formation des feuillets embryonnaires dans lequel les cellules endodermiques migrent activement hors de l’épiblaste pour atteindre leur position interne dans l’embryonDuring development, cells are progressively separated into distinct territories, delimited by embryonic boundaries. The first segregation event occurs during gastrulation, when the embryo is organised in three germ-layers, the ectoderm, the mesoderm and the endoderm. The molecular and cellular mechanisms ensuring this segregation have not yet been elucidated. During my PhD thesis, I have focused on the endoderm internalisation in the zebrafish embryo. Based on in vitro results, it has been suggested that germ-layer progenitors would be segregated by a passive cell sorting. Combining cell transplantation, live confocal microscopy and functional analyses, I have shown that endodermal cell internalisation actually results from an active migration process dependent on Rac1 and its effector Arp2/3, a direct regulator of actin. Strikingly, endodermal cells are not attracted to their internal destination but rather appear to migrate out of their neighbouring cells. This process is dependent on the Wnt/PCP pathway and N-cadherin. Furthermore, N-cadherin is sufficient to trigger the internalisation of ectodermal cells, without affecting their fate. Overall, these results lead to a new model of germ-layer formation, in which endodermal cells actively migrate out of the epiblast to reach their internal position
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Ruddell, Richard George. "An investigation into the regulatory mechanisms associated with the recombinant human 5-hydroxytryptamineâ‚â†A (5-HTâ‚â†A) receptor." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368063.
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Clowes, Robert William. "Beyond situated action : a neo-Vygotskian theory of thinking and language internalisation." Thesis, University of Sussex, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445614.
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McNee, Gavin Scott. "p38 MAP kinase regulated PLD1 activity controls stress-induced EGF receptor internalisation." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611445.
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D'Allard, Diane. "Erythropoïèse normale et pathologique, internalisation de c-Kit et morphologie du nucléole." Phd thesis, Université René Descartes - Paris V, 2013. http://tel.archives-ouvertes.fr/tel-00948122.
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L'érythropoïèse est le processus aboutissant à la production des hématies à partir d'une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d'expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l'ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu'en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l'imatinib, les érythroblastes cultivés ex vivo perdent l'expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l'érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l'imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l'imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l'imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l'imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l'imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l'imatinib lève l'auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L'étude de la taille et du potentiel prolifératif des cellules, ainsi que l'analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d'un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), " matrice " du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l'évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu'au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d'internalisation d'un récepteur à activité tyrosine kinase de classe III, c-Kit par l'imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque.
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Allard, Diane d'. "Erythropoïèse normale et pathologique, internalisation de c-Kit et morphologie du nucléole." Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05S026/document.
Full textAbstract:
L’érythropoïèse est le processus aboutissant à la production des hématies à partir d’une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d’expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l’ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu’en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l’imatinib, les érythroblastes cultivés ex vivo perdent l’expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l’érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l’imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l’imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l’imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l’imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l’imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l’imatinib lève l’auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L’étude de la taille et du potentiel prolifératif des cellules, ainsi que l’analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d’un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), « matrice » du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l’évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu’au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d’internalisation d’un récepteur à activité tyrosine kinase de classe III, c-Kit par l’imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risqueErythropoiesis is the process leading to the production of red blood cells from hematopoietic stem cell. The erythroid differentiation involves morphological cell changes, in part related to the loss of membrane expression of the type III receptor tyrosine kinase, c-Kit. In response to its ligand SCF, c-Kit is activated, then internalized and degraded by the proteasome pathway via the E3 ubiquitin ligase c-Cbl, or by the lysosomal pathway, after endocytosis. In the first part of this work, we demonstrated that in the absence of SCF and in response to tyrosine kinase inhibitor, imatinib, erythroblasts cultured ex vivo, lose membrane expression of c-Kit and accelerate their terminal differentiation. In view of these observations, we sought to understand the mechanisms involved. On an erythropoietin dependent cell line expressing c-Kit at the membrane, we showed that imatinib induces receptor internalization and degradation by the lysosomal pathway, independently of c -Cbl. Furthermore, we showed that this effect is reversible and that imatinib does not block the c-Kit re-expression after its internalization, in response to SCF. Metabolic labelling showed that imatinib does not alter synthesis or maturation of c -Kit and that the phospho-tyrosine profile of cells treated with imatinib is generally unchanged. Finally, we showed that the binding of imatinib to the catalytic pocket of c-Kit is essential for its internalization, and therefore its degradation. So, it appears that imatinib removes c-Kit self-inhibition, which seems necessary to its retention at the membrane. In the second part of this work, we studied the morphological changes of nucleoli, the site of ribosome biogenesis, during erythroid differentiation. We showed that the reduction of cell size takes place at the same time than reduction of cell proliferation and reduction of surface and volume of the Granular Compound (GC), the “matrix” of the nucleolus. Moreover, we showed by electronic microscopy, the persistence of GC at the end of maturation. Finally, we also studied the evolution of nucleoli in a pathological context of low risk myelodysplastic syndromes, which are characterized by ineffective hematopoiesis. We observed that immature pathological cells have larger GC than immature normal cells, but that during differentiation, the morphology of nucleoli is identical between normal and pathological cells. In conclusion, this work has allowed us to describe 1) the mechanisms of internalization of a class III receptor tyrosine kinase, c-Kit by imatinib and 2) the morphology of the nucleolus during normal and pathological low risk myelodysplastic syndromes of erythroid differentiation
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Natkanski, E. M. "Investigating novel components and mechanisms involved in B cell receptor-antigen internalisation." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1413024/.
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The elimination of a wide variety of infections requires the production of high affinity antibodies specific for the invading pathogen. The generation of these high affinity antibodies depends on the ability of B cells to recognise and internalise antigens from the surface of antigen-presenting cells (APCs) in an affinity-dependent manner. B cells expressing high affinity B cell receptors (BCRs) internalise, process and present more antigen, obtain better T cell help, and are selectively expanded over lower affinity equivalents. However, the molecular mechanisms by which B cells extract antigens for presentation remain unclear. Using a new fluid and flexible membrane substrate to mimic APCs, we show that B cells acquire antigen by dynamic myosin IIA-mediated contractions that pull out and invaginate the presenting membranes. Invaginations containing high affinity BCR-antigen microclusters were able to withstand the force of these contractions and recruit clathrin resulting in endocytosis. In contrast, low affinity BCR-antigen bonds quickly ruptured aborting internalisation. Thus we conclude that coupling contractility to endocytosis permits B cells to discriminate between antigen affinities, which provides a mechanism for the selective advantage seen for high affinity B cell clones in vivo. Disruptions in BCR-antigen internalisation has been linked to the growth of malignant B cells and the development of autoimmunity. Therefore, ultimately, our results could contribute to the effective design of future therapeutics for B cell diseases.
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Kirkness, Paul. "Territorial stigmatisation of French housing estates : from internalisation to coping with stigma." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8840.
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In this thesis I examine the ways in which residents of France's so-called 'banlieues' respond to everyday life in stigmatised neighbourhoods. Through a description of the processes at work in two housing estate neighbourhoods of the southern French city of Nîmes - Pissevin and Valdegour - and drawing upon an analysis of intensive interviews, I question the popular belief that residents of French banlieue-spaces come to internalise the stigmatic representations that are produced outside their place of residence. The overarching argument of the thesis is that, while it is clear that territorial stigmatisation has long-lasting and pervasive consequences for banlieue residents, affecting their sense of self and their capacity for collective action, there are a number of ways in which the 'blemish of place' is challenged and the marks of neighbourhood stigma resisted. It is important to recognise the attempts that are made within French housing estates to displace or negotiate stigmatising gazes and to confront the labels that affix themselves to place. This thesis argues that there are a variety of counter-discursive attempts to reframe and to reclaim the representations of France's housing estates that leads to the affirmation of banlieue-identities. Within the banlieues, there are solid links between residents and place, as well as between the residents themselves. Strong efforts are deployed by associations, neighbourhood committees and grassroots organisations to actively challenge the stigmatic scripts that are imposed upon stigmatised neighbourhoods. However, this thesis also draws attention to the everyday tactics that residents enact in order to cope with territorial stigmatisation and its effects. These everyday practices allow for some to cope with the heavy burden of stigma while taking control of the 'neighbourhood space'. All of these tactics challenge and 'speak back' to the labels, the stereotypes and the stigmatising language that is produced at the level of urban planning. This leads to the vital rethinking of policies that aim to displace and disperse residents in the name of social mixing, as well as urban policy initiatives that equate renovation to the demolition of housing estates within French banlieues.
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Adigbli, D. K. "A mechanistic study of photochemical internalisation and enhanced drug delivery cancer cells." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1460899/.
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Inefficient intracellular drug delivery is a significant limiting factor to success in cancer therapeutics. Photochemical internalisation (PCI) utilises the fundamental principles of photodynamic therapy (PDT): photosensitiser plus light and oxygen, at sub-lethal level to facilitate targeted intracellular drug delivery. This effect is mediated by reactive oxygen species (ROS). This thesis investigates the mechanisms underpinning sulfonated meso-tetraphenylporphine (TPPS2a) mediated PCI to enhance the delivery of two cytotoxins, saporin or mitoxantrone, and a novel Small Molecule Carrier (SMoC) in vitro. PCI of saporin was also assessed in a 3D-tumour model. In vitro experiments using 4T1 murine breast adenocarcinoma cells were performed to investigate which factors determined the likelihood of PDT versus PCI predominant cytotoxicity. The role of the intracellular REDOX environment in PDT/PCI was assessed using a free radical potentiator and quenchers. The results suggested that the localisation and total amount of ROS produced exerts the greatest influence in determining the likelihood of PDT versus PCI induced cell kill. In addition, PCI further enhanced SMoC-aided delivery of siRNA in MCF7 human breast cancer cells. A compressed collagen scaffold, embedded with 4T1 cells, was used to investigate TPPS2a-mediated PCI of saporin in a 3D tumour model. The results indicated that a 3D-model is potentially a useful tool for pre-clinical assessment of PCI. Bioluminescent PDT studies were also carried out on MCF7 cells transduced with luciferase and the 4T1-luc2 cell line, which is stably transfected with luciferase. These studies demonstrated that bioluminescence can be used to activate a photosensitiser for a cytotoxic effect (PDT). Overall, this thesis demonstrated that by further understanding the mechanisms that underpin PCI it is possible to further enhance its facilitative effects for drug delivery. The ongoing phase II clinical trials into PCI show its translational potential as a means to improve the therapeutic effectiveness of anti-cancer drugs.
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Conchon, Sophie. "Activation, desensibilisation et internalisation des recepteurs de type 1 de l'angiotensine ii." Paris 6, 1996. http://www.theses.fr/1996PA066522.
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L'angiotensine ii (angii) est impliquee dans la regulation de la pression arterielle et du metabolisme hydrosode. Cet octapeptide agit par l'intermediaire de recepteurs possedant sept segments transmembranaires (at1, at2). Chez les rongeurs, deux sous-types de recepteurs at1 (at1a et at1b) ont ete identifies. Ils sont, jusqu'a present, pharmacologiquement et fonctionnellement identiques et sont impliques dans la majorite des effetsphysiologiques de l'angii. Diverses voies de signalisation mobilisees par ces recepteurs, faisant intervenir notamment la proteine gq/11 ont ete identifiees. Ce travail concerne l'etude des mecanismes d'activation, de desensibilisation et d'internalisation des recepteurs at1. Plusieurs constructions chimeres ont ete produites, de facon a etudier l'implication de la region de jonction entre la troisieme boucle intracellulaire et le sixieme domaine transmembranaire du recepteur at1a dans la processus d'activation des voies de signalisation. L'analyse des proprietes fonctionnelles de ces mutants a revelee que ce domaine est implique differentiellement dans diverses fonctions du recepteur. D'autre part, le recepteur at1a apparait moins susceptible a l'activation constitutive que d'autres recepteurs couples aux proteines g. La liaison de l'angii induit egalement l'internalisation des complexes hormone/recepteur. Afin d'etudier le mecanisme d'internalisation de ces recepteurs, at1a, at1b et un mutant d'at1a incapable de se coupler a la proteine g ont ete exprimes en cho. Il s'avere qu'at1a et at1b sont internalises de maniere identique. De plus, les ligands peptidiques, agonistes ou antagonistes induisent l'internalisation, mais un ligand non peptidique est moins efficace. Enfin, le mutant at1a decouple est normalement internalise. Le processus de desensibilisation du recepteur at1a a ete caracterise. La desensibilisation est definie comme l'attenuation du signal apres plusieurs applications d'angii. Dans les cellules cho at1a, une desensibilisation du signal atteignant 60%, correspond a une desensibilisation homologue du recepteur a pu etre observee. Finalement, l'implication de la region c-terminale cytoplasmique du recepteur at1a dans les mecanismes d'internalisation et de desensibilisation a ete etudiee. La caracterisation de differents mutants tronques demontre qu'un domaine de sept residus est implique dans les processus d'internalisation et de desensibilisation homologue du recepteur. La deletion de cette region aboutit a un mutant presentant une reponse exacerbee a l'angii.
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Fremont, Sophie. "Internalisation épididymaire récepteur-dépendante de l'androgen binding protein testiculaire chez le rat." Nancy 1, 1988. http://www.theses.fr/1988NAN10362.
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Mise en évidence d'une immunoréactivité croisée entre l'ABP de rat et la SBP humaine. L'ABP est internalisée après liaison à la membrane des cellules principales de la tête de l'épididyme, cette liaison concerne une protéine membranaire
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Ronacher, Katharina. "Internalisation of the type II gonadotropin-releasing hormone receptor of marmoset monkey." Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/8599.
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Bibliography: leaves 102-124.The mammalian type II GnRH receptor has a C-terminal tail unlike the mammalian type I GnRH receptor, which uniquely lacks the cytoptasmic C- terminal domain. lnternalisation of a mammalian type ll GnRH receptor has never been investigated, therefore this thesis studies the internalisation pathway of the type ll GnRH receptor. As the C-terminal tail mediates rapid internalisation of many G protein-coupled receptors this research investigates the functional role of the C-terminal tail and intracellular loop in receptor internalisation. The internalisation pathway of the type ll GnRH receptor in COS-1 cells was investigated by co expressing dominant negative mutants and wild- type constructs of G protein-coupled receptor kinases (GRKs), dynamin-1 and β-arrestin 1 and 2 with the type II GnRH receptor. The results show that internatisation of the receptor requires GRK 2 and dynamin but does not require β-arrestin 1 and 2. Furthermore, inhibitors to both the caveolae pathway as well as the clathrin coated vesicle endocytosis abolished receptor internalisation indicating that both structures are involved in internalisation of the receptor. Even though in COS-1 cells the type ll GnRH receptor internatises in a β-arrestin independent manner, internalisation of this receptor can be enhanced by over-expression of wild type β-arrestin. This indicates that the type ll GnRH receptor is able to utilise a β-arrestin mediated internaltsation pathway if high levels of β-arrestin are present in the cell. The mammalian type ll GnRH receptor internalises with enhanced rate and extent compared to the tail-less human type I GHRH receptor. The role of the C-terminal tail of the type ll GnRH receptor in internalisation was investigated by measuring internalisation of C-terminally truncated mutants. It was found that the region between Gly 343 and Ser 335 within the C-terminal domain is important for receptor internalisation. Substitution of putative phosphorylation sites within this region revealed that Ser 338 and Ser 339 are critical for rapid receptor internalisation. Furthermore a serine residue in intracellular loop three (Ser 251) was shown to play a role in signalling as well as in internalisation. Since dominant negative GRK 2 could not inhibit internalisation of a mutant lacking all three serine residues, but could reduce internalisation of the wild-type receptor, we suggest that Ser 251, 338 and 339 are target of phosphorylation by GRK. However these phosphorylation sites as well as the C-terminal tail are not necessary for β-arrestin dependent internalisation. Taken together this thesis elucidates the internalisation pathway of a mammalian type lI GnRH receptor and identified residues within the C-terminal tail and intracellular loop three that are critical for rapid internalisation.
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Bailleul, Christophe. "Internalisation intraérythrocytaire d'effecteurs allostériques de l'hémoglobine : études in vitro et in vivo." Compiègne, 1990. http://www.theses.fr/1990COMPD305.
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Le transport de l'oxygène, assuré par l'hémoglobine dans les érythrocytes peut être modifie par l'internalisation d'effecteurs allostériques de l'hémoglobine. La courbe de dissociation de l'oxyhémoglobine est alors déplacée vers la droite avec augmentation de la P50. L'internalisation dans les érythrocytes de mammifères est réalisée par lyse osmotique réversible suivie d'une phase de rescellement en solution régénératrice des métabolites érythrocytaires essentiels. L'optimisation des principaux paramètres régissant l'internalisation d'inositol hexaphosphate (IHP), le plus affin des effecteurs connus de l'hémoglobine, a conduit à l'obtention d'érythrocytes charges d'IHP, aux caractéristiques intrinsèques semblables à celles des érythrocytes natifs. Une diminution de 10% du volume globulaire moyen pour une augmentation de 45 mm Hg de la P0 est observée avec un rendement cellulaire de 65%. La diminution du taux final d'ATP est corrigée par l'internalisation simultanée d'ATP. La solution d'IHP retenue est 38 mm et 250 m0sm. Les autres paramètres décisifs sont la durée ou le débit de dialyse, la concentration en hémoglobine et la température : 4°C pour la dialyse et 37°C pour le rescellement. La transfusion d'érythrocytes charges d'IHP a permis d'augmenter la P50 in vivo de façon transitoire chez le porc et la souris, et de mesurer une durée de vie érythrocytaire au moins aussi longue que celle des érythrocytes témoins. L'efficacité d'une transfusion isovolémique d'érythrocytes chargés d'IHP à hématocrite constant ou après hémodilution est caractérisée par une diminution du débit cardiaque en fonction de l'augmentation de la P50 in vivo, sans modifier la consommation d'oxygène tissulaire. L'utilisation potentielle des érythrocytes charges d'IHP en thérapeutique humaine concerne les pathologies hypoxiques ou la correction de modifications physiologiques en conditions extrêmes d'approvisionnement en oxygèneErythrocytic properties may be modified by haemoglobin effector internalization. As a consequence the oxyhemoglobin dissociation curve is shifted to the right and the P50 is increased. Internalization of allosteric effector in mammalian erythrocytes was performed using an osmotic reversible lyses and a resealing solution allowing the rejuvenation of essential erythrocytic metabolites. Inositol hexaphosphate (IHP) is the most powerful of the known allosteric effector of haemoglobin. Optimisation of the most of parameters influencing IHP internalization led to IHP-loaded erythrocytes with intrinsic characteristics closed to native erythrocytes. A 10% decrease of mean cell volume for a P50 increase of 45 torrs was measured for a cellular yield of 65%. The decrease of ATP content was corrected by simultaneous internalization of ATP. The IHP-washing solution selected was 38mM and 250 m0sm. The other parameters are the input dialysis flow or dialysis time, the haemoglobin concentration and the temperature of different steps : 4°C for dyalisis and 37°C forresealing. IHP-loaded erythrocytes transfusions induced in vivo increase of P50 in piglets and mice, with life span similar to native’s one. The isovolemic exchange-transfusion of IHP-loaded erythrocytes at constant hematocrite or under hemodilution conditions leads to a decrease of cardiac activity proportional to the in vivo P50 increase without modifying the tissular oxygen consumption. Potential uses of IHP-loaded erythrocytes in human therapy are concerned with pathological hypoxias and extreme physiological conditions of oxygen delivery
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Rosazza, Christelle. "Internalisation et trafic intracellulaire de l'ADN plasmidique délivré par électroporation in vivo." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/3067/.
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L'électroporation est une méthode physique de délivrance de molécules dans les cellules ou tissus. Le transfert des petites molécules se déroule vraisemblablement par diffusion à travers de présumés electropores alors que le transfert d'ADN plasmidique est plus compliqué et reste à élucider. Lors de l'application des impulsions électriques, la membrane devient perméable et l'ADN est électrophorétiquement poussé vers celle-ci où il est inséré en agrégats distincts pendant une dizaine de minutes. Ensuite, l'ADN est internalisé, navigue à travers le cytoplasme jusqu'à atteindre le noyau où l'expression de l'ADN est initiée. Mon travail de thèse se concentre sur l'internalisation et le trafic intracellulaire de l'ADN, deux étapes assez peu décrites dans le cadre de l'électroporation. L'internalisation de l'ADN semble se faire majoritairement par endocytose. L'utilisation d'inhibiteurs pharmacologiques de l'endocytose et de marqueurs fluorescents permet de conclure que l'endocytose médiée par la clathrine et les rafts lipidiques ainsi que la macropinocytose sont impliquées (respectivement 25%, 50% et 30%). L'endocytose semble être la principale (sinon la seule) voie par laquelle les agrégats d'ADN entrent dans les cellules. De plus amples recherches doivent être menées pour dépeindre précisément le mécanisme d'internalisation de l'ADN d'autant plus que l'endocytose médiée par les rafts lipidiques représente un large panel de possibilités (caveoline, flotilline, GEEC. . . ). Nos résultats sont en accord avec la distribution spatiotemporelle de l'ADN observée à la membrane. Une fois dans le cytoplasme, l'ADN semble suivre le trafic endosomal classique. La colocalization en dynamique effectuée dans des cellules exprimant séparément plusieurs marqueurs d'endosomes (Rab5, Rab11, Rab9 et Lamp1) démontre que l'ADN est présent dans les endosomes précoces, de recyclages et tardifs dans des proportions calculées pour être respectivement 70%, 50% et 30%, valeurs moyennées sur l'heure qui suit l'électrotransfert. Entre 1-2 h après la délivrance, 60% de l'ADN est situé dans des structures marquées Lamp1 avec une prédominance probable des lysosomes. Ces résultats sont en accord avec l'observation de l'ADN toujours présent en agrégats dans le cytoplasme et ils renforcent l'observation d'un mécanisme d'endocytose mentionné précédemment. L'ADN dans le cytoplasme est transporté activement par les filaments d'actine et les microtubules. L'utilisation d'inhibiteurs pharmacologiques combinée au suivi de particules uniques effectué sur un grand nombre d'agrégats d'ADN clairement démontre que le cytosquelette contrôle la migration de l'ADN dans le cytoplasme. L'ADN exhibe le mouvement typique des endosomes avec des phases intermittentes de transport actif et de diffusion. Les phases de transport actif ont en moyenne une vitesse de 250 nm/s, une persistance de 6 s et engendre un déplacement de 1,3 µm. Cependant, les distributions de ces paramètres sont larges avec des vitesses allant de 50 nm/s à 3400 nm/s, des déplacements de 0,1 µm à 12 µm et des durées de 2 s à 30 s. Ces résultats confirment d'autres travaux qui présentent les microtubules comme un moyen de migration de l'ADN dans les cellules. Cela est aussi en accord avec la présence de l'ADN dans des endosomes vu que leurs membranes contiennent des protéines (probablement Rabs) capables de se lier aux moteurs moléculaires. De plus, cela explique comment les gros agrégats d'ADN peuvent traverser le cytoplasme dense et atteindre avec succès le noyau, phénomène improbable par simple diffusion. Le chemin de l'ADN que nous décrivons est efficace étant donné que la perturbation d'une étape intermédiaire entraine une réduction de l'expression génique. Bien que nos découvertes doivent être confirmées et nécessitent de plus amples investigations, la prochaine étape clé à comprendre est l'échappement supposé de l'endosome qui doit forcement se réaliser afin d'obtenir une expression de l'ADNElectroporation is a physical method of delivery of molecules into cells or tissues. The transfer of small molecules most likely occurs via diffusion through putative induced electropores whereas the transfer of plasmid DNA is more complex and remains to be elucidated. During the electric field pulses, the membrane becomes permeable and the DNA is electrophoretically pushed on it where it is inserted as discrete clusters for tens of minutes. After the electric pulses, DNA is internalized, navigates through the cytoplasm until it reaches the nucleus where DNA expression is initiated. This PhD work focuses on the internalization and intracellular trafficking of the DNA, two steps rather enigmatic in the context of electroporation. The relevant cell structures that could participate into these steps are the endocytotic machinery followed by the endosomal trafficking and the cytoskeleton (actin filaments and microtubules). DNA internalization seems to be mainly performed via endocytosis. The use of pharmacological endocytic inhibitors combined with endocytic markers led us to the conclusion that clathrin- and lipid raft-mediated endocytosis as well as macropinocytosis are involved (25%, 50% and 30%, respectively). Endocytosis seems to be the main (if not the only) path by which DNA aggregates enter the cells. More investigations have to be done to depict precisely the DNA internalization mechanism especially because raft-mediated endocytosis represents a large panel of possibilities (caveolin, flotillin, GEEC. . . ) which are worth describing. These findings are in agreement with the observed spatiotemporal distribution of the DNA aggregates at the membrane (clusters of few hundreds of nm persisting for few min and inaccessible from the extracellular medium). Once inside the cells, DNA seems to follow the classical endolysosomal trafficking. Dynamic colocalization performed in cells expressing separately several labeled endosomal markers (Rab5, Rab11, Rab9 and Lamp1) allows us to conclude with no ambiguity that DNA is present in early, recycling and late endosomes in proportions calculated to be respectively 70%, 50% and 30% over the hour following the DNA electrotransfer. Between 1-2 h after delivery, 60% of the DNA is located in Lamp1-structures with most probably a predominance of the lysosomes. These results are in agreement with the observed DNA being still in clusters in the cytoplasm and it reinforced the earlier mentioned mechanism of endocytosis. DNA in the cytoplasm is actively transported by both the actin filaments and the microtubules. The use of pharmacological inhibitors combined with SPT performed on a high number of DNA aggregates clearly shows that cytoskeleton mediates the DNA journey in the cytoplasm. DNA exhibits the typical motion of endosomes with intermittent phases of active transport and diffusion. Under our experimental conditions, active motion phases features on average velocity of 250 nm/s and persistence of 6 s and leads to a displacement of 1. 3 µm. However, distributions of theses parameters are broad with velocities from 50 nm/s to 3400 nm/s, displacements from 0. 1 µm to 12 µm and persistence from 2 s to 30 s. These findings confirm previously published article pointing at the microtubules as a means of DNA migration in the cells. It is also in agreement with DNA being in endosomes since their membranes process proteins (probably Rabs) that can bind molecular motors. In addition, it explains how such big DNA clusters can travel through the highly crowded cytoplasm since successful to reach nucleus diffusion is improbable. The DNA route(s) we described correspond to the efficient one(s) since disturbance of any intermediate step results in a reduced gene expression. Although our findings must be confirmed and further investigated, the next key step to unravel is the putative endosomal escape that has to occur for successful gene expression
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Fremont, Sophie. "Internalisation épididymaire récepteur-dépendante de l'androgen binding protein testiculaire chez le rat." Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb37613669x.
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Terenzio, M. "A novel RNAi screen for neurotrophin receptor internalisation and trafficking in motor neurons." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/815424/.
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A primary focus of the Molecular Neuropathobiology laboratory is the investigation of the long range trafficking of neurotrophins and neurotrophin receptors in motor neurons (MNs). The goal of my project was to deepen our understanding of the nature of the cellular machinery controlling long-range neurotrophin trafficking in MNs, by discovering new players involved in this process. In order to achieve this goal I performed an siRNA screen in MNs derived from mouse ES cells to monitor the cell surface binding and internalization of two fluorescently tagged reporters: the binding fragment of the Tetanus neurotoxin (HC), which enters an axonal transport compartment shared with neurotrophins and their receptors, and an antibody directed against the extracellular domain of the neurotrophin receptor p75NTR. A high-throughput, lipidbased siRNA transfection method was optimised for ES cell-derived MNs and used to screen a library of siRNAs directed against a pool of genes involved in endocytosis and membrane trafficking. The primary candidate genes were subsequently validated, and one gene in particular, Bicaudal D homolog 1 (BICD1), was selected for further analyses. BICD1 is a member of the Bicaudal D family, whose members function as molecular motor adaptors with pleiotropic roles in intracellular trafficking. Bicd1 expression at E12.5 and 13.5 was restricted to the nervous system, suggesting an important role for BICD1 in neurons. I used gene-trapped ES cells to derive MNs depleted of the BICD1 protein (Bicd1gt/gt MNs), which, when challenged with either HC or the p75NTR antibody, displayed an increased intracellular accumulation of both probes. Furthermore, I found that the level of neurotrophin receptors exposed at the plasma membrane was increased in these cells compared to their wild type counterparts, suggesting that BICD1 might be involved in the regulation of neurotrophin receptor dynamics in mammalian neurons. I also found that TrkB signalling upon stimulation with the brain-derived neurotrophic factor (BDNF) in Bicd1gt/gt MNs, was impaired. This suggests that the depletion of BICD1 not only affects the trafficking of neurotrophin receptors, but also their signalling capabilities. In conclusion, this thesis work has demonstrated that the concept of high throughput screening can be applied to cells notoriously difficult to handle and transfect, such as MNs. This approach was successful in unravelling a new role for BICD1 in neurons, where it appears to regulate the intracellular trafficking and signalling of neurotrophin receptors. Taken together with the in vivo expression data, these data suggest that BICD1 plays an important role in the development and function of the nervous system.
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Laguerre, Mirella. "Création de bioréacteurs par internalisation d'enzymes dans les globules rouges humains et murins." Tours, 1990. http://www.theses.fr/1990TOUR3303.
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Bologna, Jean-Charles. "Internalisation d'oligonucléotides via une approche pro-drogue : synthèse, marquage fluorescent et pénétration cellulaire." Montpellier 2, 2001. http://www.theses.fr/2001MON20029.
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Caoduro, Cécile. "Transport d’ADN par des complexes nanotubes de carbone protamine : fonctionnalisation et internalisation cellulaire." Thesis, Bourgogne Franche-Comté, 2018. http://www.theses.fr/2018UBFCE013.
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La thérapie génique nécessite l’utilisation de vecteurs efficaces pour acheminer l’information génétique au sein des cellules cibles. Les nanomatériaux, sujet d’un grand nombre de travaux de recherche actuellement, sont ainsi devenus des candidats prometteurs au transport de gènes. Dans ce contexte, nous avons élaboré un vecteur innovant composé d’un nanotube de carbone monoparoi (puNTC) fonctionnalisé par la protamine, une protéine cationique capable de condenser l’ADN. Nous avons préparé des complexes puNTC-protamine par fonctionnalisation non covalente, et comparé à des complexes puNTC-pyrène-ammonium. Nos travaux ont permis de caractériser ces nanovecteurs et de montrer la capacité accrue des complexes puNTC-protamine à transporter l’ADN. Nous nous sommes intéressés aux différents paramètres capables d’influencer l’efficacité de transport cellulaire et avons ainsi mis en évidence une amélioration de l’internalisation cellulaire par l’utilisation de NTC oxydés et courts, associés à des fragments d’ADN de petite taille. Une étude complémentaire de modélisation a permis de corroborer et d’expliquer ces résultats, qui sont la conséquence d’une amélioration de la stabilité des complexes et d’un ratio de charge optimal offerts par les NTC courts et petits fragments d’ADN. Nous avons également étudié le mécanisme d’internalisation cellulaire de nos complexes et démontré que ces derniers traversaient la membrane plasmique par endocytose, processus rapporté par de nombreuses études tandis que d’autres décrivent un mécanisme indépendant de l’énergie. L’analyse de l’état de l’art à ce sujet nous a permis de comprendre que plusieurs mécanismes de passage membranaire sont en fait probablement intriqués, en fonction des conditions expérimentales et des types cellulaires. De part leur efficacité démontrée dans le transfert de gènes, nos complexes puNTC-protamine pourraient devenir vecteurs de piARN, petits ARN largement utilisés dans le développement de thérapies ciblées des cancersGene therapy needs efficient vectors to transport genetic information into target cells. Actuallynanomaterials have been intensively investigated and became promising candidates for genedelivery. In this way we developed a novel nanovector based on single-walled carbon nanotubes(SWCNT) functionalized by protamine, a cationic protein capable to condense DNA. We preparedSWCNT-protamine complexes via an established non covalent functionalization and compared them toSWCNT-pyrene ammonium complexes. Our study permitted to characterize these nanovectors and toshow the high efficiency of SWCNT-protamine complexes to transport DNA. We investigated theinfluence of different parameters on cellular uptake and pointed out that oxidized and short CNTassociated with short DNA fragments improved cell internalization. An additional study based onmolecular dynamic simulations allowed to confirm and understand these results. Indeed short SWCNTand small DNA fragments improve complexes stability and offer optimized charge ratio. We also studied our complexes cell internalization pathway and demonstrated that the complexes crossed theplasma membrane barrier by endocytosis, as reported by many studies while others describe anenergy-independent non-endocytotic pathway. The state of art analysis allowed us to understand thatseveral cellular uptake mechanisms are rather complementary, depending on both experimentalprocedures and cell types. Thanks to their recognized efficiency for gene delivery, our SWCNT-protamine complexes could transport piRNA, short RNA widely used in the development of targeted cancer therapy
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M'Pama-Biankouika, Marcel. "Internalisation de l'espace économique à l'exemple des multinationales françaises et la zône franc." Grenoble : ANRT, 1985. http://catalogue.bnf.fr/ark:/12148/cb37594994p.
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Hislop, James Nicholas. "An investigation of the relationship between gonadotrophin-releasing hormone receptor structure, internalisation and signalling." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368565.
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Zimmermann-Meisse, Gaëlle. "Internalisation des leucotoxines de S. aureus dans les cellules cibles et conséquences cellulaires associées." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ113/document.
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S. aureus sécrète de nombreux facteurs de virulence qui lui permettent de lutter efficacement contre le système immunitaire, afin de favoriser la dissémination de la bactérie dans l’organisme hôte. Parmi ces molécules, les leucotoxines ciblent principalement les cellules myéloïdes comme les neutrophiles, les macrophages ou encore les monocytes, et sont formées par deux sous-unités : une de classe S et une de classe F. La Leucodine de Panton et Valentine (LPV) et l’Hémolysine γ HlgC/HlgB sont deux leucotoxines dont le composant de classe S se fixe sur l’un des récepteurs du système du complément, le C5aR. Naturellement activé par l’anaphylatoxine C5a, le C5aR voit son activité modifiée lors d’une interaction avec la LPV ou HlgC/HlgB, tout du moins pour la libération du calcium intracellulaire. Ces deux leucotoxines, à l’instar du C5a, sont internalisées dans le neutrophile humain et utilisent le transport rétrograde pour atteindre l’appareil de Golgi. Elles peuvent rester dans la cellule jusqu’à 3h sans susciter la mort pour le neutrophile. Plus tard, à 6h, seule la LPV induit de l’apoptose et de la NEToseS. aureus secretes many virulent factors which allow to efficiently fight the immune system, in a way to promote the bacterial spreading inside the host. Among these molecules, the leukotoxins target myeloid cells such as neutrophils, macrophages and monocytes, and are composed of two subunits: one of class S and one of class F. Panton and Valentine Leukocidin (PVL) and γ-Haemolysin HlgC/HlgB are two leukotoxins whose S-component binds to the C5aR, one of the complement system receptors. Naturally activated by the C5a anaphylatoxin, the activity of the C5aR is modified by the PVL and HlgC/HlgB interaction, for the intracellular calcium release. These two leukotoxins, as C5a, are internalised inside the human neutrophils and use the retrograde transport to reach the Golgi apparatus. These can rest inside the cells until 3h without neutrophil dead. Later, at 6h, only PVL induces apoptosis and NETosis
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Rezaï, Xavier. "Activation et internalisation du récepteur aux opiodés delta en tranche aiguë hippocampique de souris." Strasbourg, 2011. https://publication-theses.unistra.fr/public/theses_doctorat/2011/REZAI_Xavier_2011.pdf.
Full textAbstract:
Le récepteur aux opioïdes δ fait partie du système opioïde, un système neuromodulateur compose de peptides endogènes (enkephalines, dynorphines et beta--‐endorphine) et de trois récepteurs (μ, δ et κ). Notre laboratoire s’intéresse aux réponses adaptatives des récepteurs aux opioïdes à l’usage abusif des drogues. Le récepteur δ est exprimé dans l’hippocampe, siège de la mémoire épisodique, et son implication dans les interactions entre drogue et contexte d’administration fait l’objet d’un intérêt croissant. Bien qu’identifies de longue date, l'expression et le rôle de ces récepteurs dans les interneurones de l'hippocampe restent peu étudiés, tout comme la localisation et la fonction de ces derniers au sein des différents réseaux hippocampiques, en particulier le CA1. Par ailleurs, la majorité des études portant sur le récepteur δ ont été réalisées en systèmes hétérologues ou en cultures primaires de neurones. Si ces modèles ont permis de comprendre les mécanismes de base du fonctionnement de ce récepteur, ils ne prennent pas en compte la diversité des types neuronaux. De plus, la morphologie et la Physiologie des neurones isoles sont vraisemblablement modifiées par rapport à la situation in vivo. Il apparait donc primordial d‘aborder l’étude de ce récepteur en réseau intégré afin d'appréhender l'activation et l'internalisation du récepteur δ dans le contexte le plus physiologique possible. Pour aborder ces questions, j’ai utilisé une souris knock--‐in DOR--‐eGFP développée au laboratoire, qui exprime en lieu et place du récepteur δ natif, une version rendue fluorescente par couplage a son extrémité C terminale de la protéine verte fluorescente (eGFP). Cette souris est un excellent outil pour visualiser in vivo. [etc. . . ]The delta opioid receptor belongs to the opioid system, a neuromodulatory system composed of endogenous peptides (enkephalins, dynoprhins and beta-endorphin) and of three receptors (mu, delta and kappa). Our laboratory is interested in opioid receptors adaptative responses to drugs of abuse. The delta opioid receptor is expressed in the hippocampus, and the important role of this brain structure in episodic memory brings growing interest toward it’s implication in drug / context interactions. Although the expression of the delta receptor in hippocampal interneurons is known for a long time, it’s function in hippocampal networks such as CA1 remains poorly studied. Moreover, most studies focusing on the delta receptor were performed in heterologous systems or primary neuron cultures. These models have helped understanding the basic functioning of this receptor, but they do not take into account the diversity of neuronal types. In addition, the morphology and physiology of single neurons in culture are likely to change from the in vivo situation. It is therefore important to study of this receptor in an integrated network in order to understand activation and internalization of the delta receptor in a physiological context. To address these questions, I used a knock-in mouse (DOR-eGFP) developed in the laboratory, that express a fluorescent version of the delta receptor made by adding a green fluorescent protein (eGFP ) at the C-terminus of the receptor. This mouse is an excellent tool to visualize the receptor with subcellular resolution in vivo and thus compensate for the lack of specific antibodies. [etc. . . ]
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Dorval, Jérémie. "La taxe sur l'essence et internalisation des coûts sociaux des véhicules légers au Québec." Master's thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/26380.
Full textAbstract:
Tableau d'honneur de la Faculté des études supérieures et postdorales, 2015-2016L’utilisation des véhicules légers crée plusieurs externalités qui sont supportées par la société. Qu’elles soient liées à la congestion, aux accidents de la route, à la pollution locale ou au réchauffement global, ces externalités engendrent une défaillance du marché puisque les automobilistes ne supportent pas la totalité des coûts qu’ils génèrent. Pour pallier à cette lacune, ce mémoire propose d’utiliser la taxe sur l’essence, comme solution de second rang, afin d’internaliser les coûts sociaux. Nos résultats suggèrent que la taxe d’accise actuelle de 0,292$/L est suffisante pour internaliser les coûts externes présents en milieu non-urbain, soit ceux liés aux accidents, à la pollution locale et au réchauffement global. Cependant, il faudrait l’augmenter à 1,53$/L dans la Région Métropolitaine de Recensement de Montréal et à 1,18$/L dans celle de Québec afin d’internaliser également les coûts externes de congestion. Ces ajustements permettraient de réaliser un gain de bien-être de 824,7 M$ pour le Québec et génèreraient des revenus additionnels pour l’État de plus de 2,5 G$.
The use of light duty vehicle is responsible for many externalities borne by society. Either they are due to congestion, road accident, local pollution or global warming, there is disequilibrium because car users do not support the entire cost of driving. To address this loophole, this master’s thesis suggests the use of gasoline tax, as a second best solution, to internalize these social costs. Our results suggest that the actual excise tax of 0.292$/L is appropriate to internalize the externalities into non-urban areas, which are related to road accident, local pollution and global warming. However, they suggest that the actual excise tax should be higher to internalize the external cost of congestion in urban areas. In fact, the excise tax in the Census Metropolitan Area of Montréal and Québec should be 1.53$/L and 1.18$/L respectively. These adjustments could create a welfare gain of 824.7M$ for the Province of Québec with an increase in tax revenues of over 2.5G$.