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Coppola, Stefano, Daniela Pozzi, Giulio Caracciolo, and Thomas Schmidt. "Intracellular trafficking of lipoplexes." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-182512.
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Guise, Christopher Paul. "The intracellular trafficking of ricin." Thesis, University of Warwick, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392938.
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Liu, Xiao Li. "Nephrin: cellular trafficking and intracellular interactions /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-899-8/.
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McKay, Jodi Ho-Jung. "HRas intracellular trafficking and signal transduction." [Ames, Iowa : Iowa State University], 2007.
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Cotta, Doné Stefania. "Nephrin - intracellular trafficking and podocyte maturation /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-411-2/.
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Tonn, Daniela. "Intracellular trafficking of Leishmania major peptidases." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/1649/.
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Leishmania resides inside mammalian macrophages, from where it is thought to manipulate the host immune system by releasing virulence factors. The cysteine peptidase CPB has been shown to be secreted by the parasite and act as such a virulence factor. CPB is released through the flagellar pocket while being trafficked to the lysosome. Thus, in this project, the intracellular localisations of eight other L. major peptidases were analysed by fluorescence microscopy, after tagging the enzymes with green fluorescent protein (GFP). The candidate peptidases were chosen by bioinformatics analyses and predictions of N-terminal secretory signal peptides and potential transmembrane domains. The aim was to find a peptidase accumulating in the flagellar pocket of the cell, from where it could be secreted. Five candidate peptidases (a ubiquitin hydrolase, a CaaX prenyl protease, a zinc carboxypeptidase and two rhomboid peptidases) localised to the mitochondrion, which was unexpected. Another, a calpain-like peptidase, localised to the flagellum but not to the flagellar pocket. A serine carboxypeptidase was found very close to the flagellar pocket, possibly in small vesicles budding off or fusing with the pocket membrane, but did not co-localise with a flagellar pocket marker. The bioinformatics predictions differed from the experimental results here and, additionally, using different algorithms to predict protein properties resulted in contradictory predictions in several cases. This suggests that generic protein prediction programmes for mammalian or higher eukaryotic proteins can be unreliable and of limited usefulness for Leishmania proteins. This corroborates the notion that Leishmania may use novel, non-classical secretory pathways rather than or in addition to those characterised for higher eukaryotes. The L. major Bem46-like serine peptidase (LmjF35.4020) of the Clan SC (Family S9) was the only candidate peptidase that localised to the flagellar pocket when labelled with GFP. This was an indication that this enzyme may be released from the cell and could act as a virulence factor. Alternatively, it may be a resident protein of the flagellar pocket. Deleting the Bem46 gene in L. major did not have a measurable effect on promastigote growth or on footpad lesion development in mice inoculated with Bem46-deficient cells, so it does not appear to play a role as a major virulence factor. Apart from the secretion of virulence factors, rapid protein turnover, e.g. in the lysosome, is important for the infectivity of Leishmania. To investigate lysosome structure and function in L. major, a potential LMP (lysosomal membrane protein) was identified by bioinformatics. Thus far, no resident membrane proteins of the Leishmania lysosome are known and identifying such a protein would provide a useful marker for the closer investigation of this important organelle. In this project, the location and role of the LMP protein LmjF30.2670 was investigated using GFP-tagging and fluorescence microscopy. The experiments showed that LMP is not lysosomal in L. major, rather, it could be observed localising to a distinct, elongated and sometimes doughnut-shaped structure in close proximity to the kinetoplast. This structure was not directly associated with the flagellar pocket or the cell membrane, its position in the cell was variable within a certain area alongside the kinetoplast, it appeared to duplicate during cell division and it did not co-localise with the endocytic / lysosomal marker FM4-64. Deletion of the LMP gene did not have any effect on promastigote growth in cell culture and only a small and transient slowing effect on the development of mouse footpad lesions after inoculation with LMP-deficient L. major. Lysosomal membrane proteins can be targeted to the lysosome by the protein carrier complex AP3, which binds to tyrosine or dileucine motifs in cargo proteins. LMP contains two such tyrosine motifs at its C-terminus, but disruption of these by site-directed mutagenesis did not affect LMP localisation, suggesting that its trafficking is AP3-independent, which is in accordance with the non-lysosomal localisation of LMP. Finally, the lysosome-like acidocalcisome organelles have previously been shown to rely on the protein carrier complex AP3 for normal structure and function. In AP3-deficient Leishmania, the acidocalcisomes are defective and, at the same time, parasite virulence is markedly reduced (Besteiro et al., 2008o). To analyse how AP3 is important for acidocalcisome morphology and function, a proton pump of the acidocalcisomal membrane, the V-H+-PPase, was investigated by GFP-labelling and fluorescence microscopy. In wild type L. major the V-H+-PPase could be shown to localise to the acidocalcisomes, whereas in AP3-deficient cells it was not detectable, suggesting that the protein is mislocalised and likely degraded. The V-H+-PPase also contains several tyrosine motifs that may interact with AP3. The two most prominent of these were disrupted by site-directed mutagenesis, but this did not affect the localisation of the V-H+-PPase, suggesting that these two sites are not, or not solely, important for AP3 binding or that the V-H+-PPase is not bound by AP3 directly.
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Wilson, Rona Kirstin. "Intracellular pathways in prion peptide trafficking." Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433105.
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Mukhtar, Mohammed. "Regulation of intracellular trafficking of glucokinase." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419994.
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Henaff, Daniel. "Adenovirus biology : receptors and intracellular trafficking." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20138/document.
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Les adénovirus ont une double nature, soit comme pathogène omniprésent qui peuvent occasionnellement causer des maladies soit comme vecteurs utilisés de transfert de gène. À nos connaissances, les 30 premières minutes depuis la liaison au récepteur jusqu'à l'arrivée au pore nucléaire sont identiques pour le pathogène comme pour le vecteur. L'objectif de ma thèse était de comprendre les mécanismes impliqués dans la liaison au récepteur, l'internalisation, l'échappement et le trafic endosomal vers le MTOC. J'ai d'abord étudié le mécanisme impliqué dans l'hémagglutination des virus à tropisme pour CAR et à tropisme pour SA. J'ai identifié la présence de CAR sur les érythrocytes humains et montré qu'il était le principal responsable de l'agglutination induite par les virus à tropisme pour CAR. De plus, j'ai montré que la présence de CAR sur les érythrocytes pouvait piéger le virus dans le sang et ainsi empêcher l'infection au niveau du foie. Dans un deuxième temps, j'ai participé à la caractérisation du rôle de la protéine VI et la translocation du virus au MTOC. Nous avons montré que Nedd4 était impliqué dans le ciblage du virus au MTOC via l'ubiquitination de la protéine VI. Enfin, j'ai travaillé sur le neurotropisme de CAV-2 et caractérisé sa localisa tion subcellulaire au niveau des synapses. J'ai montré qu'une partie de CAR était localisée dans des radeaux lipidiques à la synapse et que CAV-2 entrait via la voie de recyclage des vésicules synaptiquesAdenoviruses have a dual nature as ubiquitous pathogens that occasionally cause life-threatening disease and their use as gene transfer vectors. To the best of our current knowledge, the first 30 min from binding to nuclear pore docking of both wild-type virus and vector are identical. The goal of my thesis is to understand different mechanisms involved in receptor binding, internalization, endosomal escape and trafficking to the MTOC. First I studied the mechanism involved in hemagglutination of CAR-tropic and SA-tropic viruses. I identified the presence of CAR on human erythrocytes and showed that it was the main responsible for the agglutination mediated by CAR-tropic viruses. Moreover, I show that CAR on erythrocytes can sequester virus in the bloodstream and block liver infection. In a second part I participated to the characterization of the role of the protein VI and the translocation of HAd to the MTOC. We showed that Nedd4 was involved in the targeting of the virus to MTOC through ubiquitination of this protein VI. Finally, I worked on the neurotropism of CAV-2 and characterize its subcellular localization at the synapse. I showed that a part of CAR was localized in lipid raft at the synapse and enter through the synaptic vesicle-recycling pathway
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Hutchinson, James Lawrence. "Salmonella interactions with host intracellular trafficking pathways." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611631.
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Xiao, Wu. "Intracellular trafficking of adeno-associated virus type 2." [Gainesville, Fla.] : University of Florida, 2002. http://purl.fcla.edu/fcla/etd/UFE1001195.
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Nesbeth, Darren Nicholas. "Biochemical studies of intracellular trafficking pathways in eukaryotes." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313830.
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luheshi, nadia m. "the intracellular trafficking and actions of interleukin-1." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492854.
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Inflammation is vital for host defence against infections and tissue injury. However, dysregulated inflammation contributes to the pathogenesis of major peripheral and central nervous system (CNS) diseases. lnterleukin-1 (IL-1) is a key proinflammatory cytokine, and is implicated in the pathogenesis of many of these diseases.
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Copier, John Paul. "Investigation of the intracellular trafficking of HLA-DM." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321964.
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Kershaw, Tom. "On the intracellular trafficking of CC chemokine receptor 5." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444245/.
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CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) involved in the recruitment of a subset of leukocytes to sites of inflammation. In addition, CCR5 functions as a co-receptor for several strains of the human immunodeficiency virus (HIV). The finding that agonist-induced internalisation of CCR5 can protect susceptible cells from infection in vitro has stimulated research demonstrating that activated receptors are phosphorylated on C-tail serine residues and bind p-arrestins, which couple them to the clathrin-mediated endocytic machinery. After internalisation, receptors are recycled to the cell surface via a perinuclear recycling compartment, identified as the recycling endosome. Here, I extend these findings, demonstrating that CCR5 also traffics through the trans- Golgi network (TGN), and thus, the perinuclear recycling compartment can be considered to comprise both recycling endosome and TGN elements. Moreover, I show that Rabll regulates trafficking through this compartment and that dynamin inhibition blocks the recycling of CCR5. In combination with other electron microscopy studies, my data support the notion that clathrin and dynamin are involved in the recycling of CCR5 from the recycling compartment. Through morphological and biochemical analysis, I also show that p-arrestins maintain an interaction with CCR5 into the recycling compartment. Kinetic analysis of receptor recycling suggests that p-arrestin2 acts a negative regulator of CCR5 recycling but my data do not discount the possibility that p-arrestins couple CCR5 to clathrin to effect recycling. In keeping with the sustained p-arrestin interaction, CCR5 molecules remain phosphorylated as they traffic to the recycling compartment, but contrary to previous reports showing that receptor C-tail phosphorylation is required for high-affinity p-arrestin binding, I show that a phosphorylation-deficient CCR5 mutant undergoes p-arrestin-dependent agonist-induced internalisation. In addition to its relevance for HIV biology and inflammation, this study contributes to an understanding of GPCR trafficking in general.
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Cebrián, José Ignacio. "Intracellular trafficking during antigen cross presentation in dendritic cells." Paris 5, 2011. http://www.theses.fr/2011PA05T045.
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Les cellules dendritiques (DCs) jouent un rôle essentiel dans l’initiation des réponses immunitaires adaptatives par les lymphocytes T. Ces cellules présentent des antigènes phagocytés sur les molécules de classe I et II du complexe majeur d’histocompatibilité (CMH) pour activer les lymphocytes T CD8+ et CD4+, respectivement. Contrairement à la présentation restreinte aux molécules de classe II du CMH, les voies intracellulaires impliquées dans la présentation des antigènes internalisés sur des molécules du CMH de classe I, un processus appelé présentation croisée, sont encore largement inconnus. Les DCs sont les seules cellules présentatrices d’antigènes à effectuer efficacement la présentation croisée. Ce processus a été impliqué dans l’établissement des réponses immunitaires cytotoxiques contre des bactéries, des tumeurs et certains virus qui n’infectent pas les DCs. Parmi les nombreuses caractéristiques, qui ont été proposées comme étant importantes pour la présentation croisée, la présence de composants du réticulum endoplasmique (RE) dans les phagosomes a généré une grande controverse. Ceci est en partie dû au fait que les mécanismes par lesquels ces composants sont recrutés dans la voie d’internalisation pour rencontrer les antigènes sont inconnus. En particulier, il reste à déterminer si des évènements de fusion entre le RE et les phagosomes ont lieu et s’ils sont nécessaires à la présentation croisée. Nos résultats ont identifié Sec22b comme un régulateur clé de la présentation croisée et des fonctions phagosomales dans les DCs. Cette molécule qui appartient à la famille des protéines SNARE est localisée dans le compartiment intermédiaire RE-Golgi et s’associe avec la Syntaxine 4, une SNARE de la membrane plasmique, qui est présente dans les phagosomes. La déplétion de Sec22b inhibe le recrutement de protéines résidentes du RE vers le phagosome et vers la vacuole contenant le parasite Toxoplasma gondii. Cette déplétion inhibe également la présentation croisée par les DCs d’antigènes phagocytés, endocytés, ou exprimés par T. Gondii ou E. Coli dans les DCs infectées par ces pathogènes. De plus, dans les DCs Sec22b déficientes, dans lesquelles les phagosomes sont dépourvus de composants du RE, la maturation phagosomale est accélérée et l’activité protéolytique est augmentée. L’ensemble de nos résultats montre que la fusion entre le RE et les phagosomes régule les fonctions de la voie d’internalisation des antigènes de manière à la rendre compatible et efficace pour leur présentation croiséeDendritic cells (DCs) are essential players in the initiation of adaptive T cell-mediated immune responses. They present phagocytosed antigens on class I and II molecules of the Major Histocompatibility Complex (MHC) to activate CD8+ and CD4+ T lymphocytes, respectively. In contrast to MHC class II-restricted presentation, the intracellular pathways involved in the presentation of internalized antigens on MHC class I molecules, a process known as cross presentation, remain unclear. DCs have been shown to be the only antigen presenting cells that can perform efficiently antigen cross presentation, and this process has been involved in establishing cytotoxic immune responses against bacteria, tumours and certain viruses that do not infect DCs. Among the many features that have been proposed to be important for cross presentation, the presence of endoplasmic reticulum (ER) components in phagosomes has generated a big controversy in the field. This is in part due to the fact that there is no evidence about the mechanism by which these components are recruited to the internalization pathway to encounter the antigens. Also, it is not clear if such fusion between phagosomes and the ER is absolutely required for cross presentation. Here we identify Sec22b as key regulator of cross presentation and of phagosomal functions in DCs. This protein that belongs to the SNARE family localizes to the ER-Golgi intermediate compartment and pairs to the plasma membrane SNARE Syntaxin 4, which is present in phagosomes. Depletion of Sec22b in DCs inhibits the recruitment of ER-resident proteins to phagosomes and to the vacuole containing the Toxoplasma gondii parasite. In Sec22b silenced DCs, cross presentation is compromised after antigen phagocytosis or endocytosis, as well as after invasion by T. Gondii or infection by E. Coli. We also observed that ER-deficient phagosomes acquire more rapidly lysosomal markers and display a higher proteolytic activity than normal DC phagosomes. Our results suggest that the fusion of the ER to phagosomes is essential for cross presentation not only by contributing with the MHC class I presentation machinery, but also by delaying phagosome maturation and promoting cross presentation conditions
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Pires, Liliana Raquel Fernandes. "Chitosan gene delivery : from intracellular trafficking to gene expression." Master's thesis, Universidade de Aveiro, 2007. http://hdl.handle.net/10773/2250.
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Mestrado em Engenharia Biomédica - BiomateriaisO quitosano é um policatião de origem natural que tem vindo a ser investigado como sistema não viral de vectorização de genes devido à sua biocompatibilidade e baixa toxicidade. No entanto, a sua baixa eficiência de transfecção tem dificultado o seu uso generalizado. Num estudo anterior mostrámos que a conjugação de resíduos de imidazol a cadeias de quitosano resulta numa melhoria da eficiência de transfecção do polímero. O principal objectivo deste estudo foi avaliar a aplicação de quitosano modificado com imidazol (CHimi) como vector para entrega de genes em medicina regenerativa, bem como encontrar novas vias para melhorar a eficiência. A expressão genética mediada por CHimi com dois graus de substituição - 13% e 22% das aminas primárias do quitosano - foi avaliada em células 293T (células embrionárias humanas do epitélio do rim) por um período de 8 dias, usando o gene da β-Galactosidase (β-gal) como gene repórter. Os complexos de CHimi-DNA foram preparados numa razão molar de 18 entre aminas primárias e grupos fosfato. As células transfectadas com estes complexos apresentam um pico de actividade da β-gal às 72 horas pós-transfecção, verificando-se a expressão sustentada da proteína repórter durante todo o período de avaliação. Nestas condições a viabilidade celular não é comprometida. Quando se efectua um segundo tratamento das células com complexos à base de CHimi, a actividade de transfecção volta a aumentar, sem haver alterações na viabilidade celular. Verificou-se também que células transfectadas com estes vectores sobrevivem a um ciclo de congelação/descongelação, mantendo uma actividade de transfecção sustentada no tempo. Uma polietilenimina comercial (Escort V) foi usada como referência neste estudo. Apesar de os níveis de transfecção mediados por este vector serem duas ordens de grandeza mais elevados, a viabilidade celular decresce até aos 50% após cada tratamento. De forma a investigar o processo de transfecção mediado por polímeros de CHimi, o tráfego intracelular destes complexos foi estudado por microscopia confocal de varrimento laser. Complexos de CHimi e DNA marcados com fluoróforos foram encontrados no citoplasma celular 2 horas depois da transfecção, sendo detectados até 48 horas pós-transfecção. Estes resultados podem em parte explicar a expressão sustentada de β-gal ao longo do tempo. Os complexos foram detectados no interior do núcleo 4 horas pós-transfecção. O DNA marcado com fluorescência não foi observado na forma livre em nenhum dos momentos analisados, enquanto que CHimi foi detectado num evento único no citoplasma. Num ensaio “cell-free” de transcrição/tradução in vitro não foi detectada a síntese de proteína quando o DNA estava complexado com CHimi, apesar de este ser expresso na ausência do polímero. Este conjunto de resultados sugere que, apesar dos complexos poderem ser encontrados no interior do núcleo rapidamente após a transfecção, a expressão genética parece depender da desintegração do complexo. O CHimi é um potencial candidato a vector para transporte de genes num cenário de regeneração. Este material medeia uma expressão proteica sustentada sem afectar a viabilidade celular. Com este sistema, as células toleram uma segunda adição de complexos, pelo que a administração repetida poderá ser potencialmente usada como estratégia para prolongar o efeito terapêutico de uma proteína de interesse. Em relação aos resultados de tráfego intracelular dos complexos, e considerando o perfil de expressão genética obtido, pode pôr-se a hipótese de que a expressão sustentada do gene resulta de um processo de libertação dependente do tempo. Neste sentido, ajustar a velocidade de degradação dos polímeros de CHimi pode ser usado como estratégia para melhorar o processo de expressão do gene tendo em vista o fim terapêutico pretendido. ABSTRACT: Chitosan is a polycation of natural origin, emerging in the non-viral gene delivery vectors scene due to its biocompability and low cytotoxicity. However, its low transfection efficiency has hampered its wide application so far. We have previously shown that grafting imidazole moieties into the chitosan backbone results in improved transfection efficiency of this polymer. The main goal of this study was to assess the application of imidazole-grafted chitosan (CHimi) as gene delivery vector in a regenerative medicine scenario and to find avenues to further improve its efficiency. Gene expression mediated by CHimi with two degrees of substitution - 13% and 22% of chitosan primary amines - was assessed in 293T cells for periods up to 8 days, using the β-Galactosidase (β-gal) gene as reporter gene. CHimi- DNA complexes were prepared at a primary amine to phosphate groups molar ratio of 18. Cells transfected with the CHimi-based complexes have a peak of β-gal activity 72 hours post-transfection and show a sustained β-gal production for 8 days. During this time period cell viability is not impaired. When a second treatment with CHimi-based complexes is performed, transfection activity increases, without changes on cell viability. Additionally, cells transfected with CHimi-based vectors are able to withstand a freeze/thawing cycle, maintaining a sustained transfection activity. A commercially available polyethylenimine (Escort V) was used as a reference. Though transfection levels are two orders of magnitude higher, cell viability decreases up to 50% after each treatment. In order to investigate the transfection process mediated by CHimi-based vectors a study of the intracellular pathway of the complexes has been performed by confocal laser scanning microscopy. Complexes formed by fluorescently labeled CHimi and DNA were found inside the cell cytoplasm 2 hours after transfection and were detected up to 48 hours post-transfection. These results could explain in part the sustained gene expression over time. Complexes were detected inside cell nucleus since 4 hours post-transfection. Fluorescently labeled DNA in the free form was not observed at any of the time points analyzed. Free CHimi was detected in the cytoplasm in an atypical event. In a cell-free in vitro transcription/translation assay no protein production was detected when DNA was complexed with CHimi, though expressed when using plasmid DNA in the absence of CHimi. Taken together these results suggest that, though CHimi-based complexes can be detected inside cell nucleus promptly after transfection, gene expression is dependent on the complex disassembling. CHimi is a potential candidate vector for gene delivery in a regenerative scenario. This material is able to mediate a sustained protein expression without impairing cell viability. In our system, cells can sustain another addition of the complexes suggesting that repeated administration could be used as a strategy to prolong the therapeutic effect. In view of the trafficking results and considering the gene expression profile, one can hypothesize that the observed sustained transgene expression is a time dependent release process. Thus, tuning the degradation rate of CHimi-based polymers could be a strategy to further improve the overall transgene expression process to fulfill the therapeutic end.
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Stévenin, Virginie. "Epithelial cell invasion and intracellular trafficking triggered by Salmonella Typhimurium." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC267.
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La Salmonellose est une maladie gastro-intestinale d’origine alimentaire contractée par plusieurs dizaines de millions de personnes chaque année. Bien que généralement bénigne, cette maladie engendre encore aujourd’hui des milliers de morts par an, en particulier chez les jeunes enfants et les personnes immunodéprimées. Au cours de cette thèse, nous avons disséqué les stratégies mises en place par la bactérie Salmonella lors de l’infection de cellules épithéliales. Salmonella est dotée de flagelles qui lui permettent de se déplacer à la surface des cellules de l’intestin et de cibler certaines cellules hôtes à infecter. Les critères de sélection de la bactérie étaient jusqu’à présent méconnus. Nous avons combiné une approche in vitro avec des outils de modélisation, afin de prédire mathématiquement les chances pour une cellule épithéliale d’être infectée ou non. Nous avons ainsi démontré que les salmonelles sont capables de sélectionner certaines cellules à infecter en fonction de leur microenvironnement local. Qui plus est, l’invasion d’une cellule hôte par Salmonella augmente les chances de cette cellule d’être infectée par d’autres salmonelles, révélant un mécanisme de coopération au long-terme entre salmonelles lors d’une infection. Pour pénétrer au sein de sa cellule hôte, Salmonella induit la formation d’expansions membranaires et s’engouffre dans une vacuole intracellulaire appelée SCV pour “Salmonella-Containing Vacuole”. Les SCVs peuvent soit se développer en une niche vacuolaire réplicative, soit se rompre, entraînant la libération de Salmonella dans le cytosol. La nature précise du compartiment d’entrée de Salmonella est demeurée longtemps débattue. En combinant des techniques de microscopie optique et électronique, nous avons révélé que Salmonella entre dans les cellules épithéliales dans une vacuole “étroite”, entourée de larges macropinosomes formés au site d’infection. La formation de ces macropinosomes dépendant de l’infection bactérienne, nous avons renommé ces compartiments “IAMs” pour “Infection-Associated Macropinosomes”. Quelques minutes après leur formation, les IAMs fusionnent avec la vacuole de Salmonella, permettant un rapide élargissement de la vacuole bactérienne. L’absence de fusion entraine quant à elle, la rupture de la vacuole bactérienne et la libération de Salmonella dans le cytosol. Ainsi, l’interaction entre les IAMs et la vacuole de Salmonella détermine les conditions de vie intracellulaire de la bactérie. Afin d’identifier les facteurs hôtes impliqués dans ce processus, nous avons développé une méthode d’isolation spécifique des IAMs et identifié les protéines qui les constituent. En particulier, nous avons révélé la présence de plusieurs protéines SNAREs pouvant permettre la fusion entre les IAMs et la vacuole bactérienne. Cette fusion est suivie d’une perte de membrane de la SCV via la formation de tubules membranaires. Nos résultats suggèrent que le mécanisme de rupture dépend de l’équilibre entre le gain et la perte de membrane apportés réciproquement par la fusion avec les IAMs et la formation de tubules.Ensemble, ces travaux nous ont permis d’élucider de nouvelles étapes clefs du mécanisme d’infection intracellulaire de SalmonellaSalmonellosis is a foodborne gastroenteritic disease causing tenths of millions of cases per year. While the disease is commonly benign, it still leads today to hundreds of thousands of deaths per year, especially in young children and immunodepressed people. Along this Ph.D., we dissected the molecular and cellular strategies of Salmonella during its infection of epithelial cells. Thanks to its flagella, Salmonella can move on the surface of intestinal cells and target specific host cells for infection. The selection criteria of the bacteria have been mostly unknown so far. We set up a pipeline of automatic acquisition and image analysis combined with mathematic modeling to forecast the probability of a given epithelial cell to be infected or not. We demonstrated that Salmonella can target specific host cells depending on features of their local environment, such as the localcell density. Besides, we found that the infection of an epithelial cell by Salmonella directly increases the vulnerability of the same cell to be reinfected, demonstrating the existence of a mechanism of long-term cooperation between bacteria.To penetrate targeted cells, Salmonella induces membrane ruffles leading to its engulfment into a membrane-bound compartment. This compartment can maturateand form a replicative vacuolar niche called the “Salmonella-Containing Vacuole”(SCV). Alternatively, the bacteria can rupture its vacuole leading to Salmonella release into the cytosol. A discrepancy existed in the field regarding the nature of theearly Salmonella-containing compartment. Coupling live fluorescence microscopy with correlative light electron microscopy, we revealed that Salmonella enters inepithelial cells in a tight compartment distinct from the surrounding macropinosomes formed at the infection site. As the bacterial infection induces the formation of those macropinosomes, we termed them “Infection-Associated Macropinosomes” (IAMs). Few minutes post-infection these endomembrane compartments can fuse with the SCV leading to its enlargement. Besides, the absence of fusion results in SCV destabilization and vacuolar escape. Thus, the interaction between IAMs and SCV determine the lifestyle of Salmonella.To identify the host factors driving SCV-IAM interactions, we developed a highlyspecific fractionation method to isolate IAMs. After proteome analysis by massspectrometry, we revealed the recruitment of specific SNAREs at the IAMs that couldallow SCV-IAMs fusion. Concomitantly to SCV-IAM fusions, we also monitored theloss of portions of the SCV membrane through the formation of membrane tubules.Our results suggest that the rupture mechanism depends on the equilibrium between the gain and the loss of membrane at the SCV via IAM fusions and tubular extraction, respectively. Together, our work provides a new comprehensive model of the early steps of Salmonella invasion of epithelial cells
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Berka, Ursula, Martin Volker Hamann, and Dirk Lindemann. "Early Events in Foamy Virus - Host Interaction and Intracellular Trafficking." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-127078.
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Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.
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Gupta, Neetu. "Inhibitors of intracellular trafficking active against plant and bacterial toxins." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112328.
Full textAbstract:
Les toxines Shiga (Stx) sont produites par Shigella dysenteriae et certaines espèces d’E. coli transmisent aux humains par la consommation d'aliments contaminés et causant des maladies graves. La toxine Stx est libérée par les bactéries dans l'intestin et par la suite, traverse les vaisseaux sanguins en aval pour atteindre leurs principaux organes cibles, notamment les reins. Les dommages causés aux reins peuvent entraîner des complications graves notamment Le syndrome hémolytique urémique (SHU). A ce jour, il n’existe aucun traitement disponible contre le SHU. Les toxines Stx usent du transport rétrograde intracellulaire pour infester les cellules endothéliales rénales et atteindre leur cible cytosolique, l'ARN ribosomal 28S. Via un screening à haut débit, il a été démontré que le composé Rétro-2 bloque le trafic rétrograde de Stx à l'interface Endosome-TGN, sans affecter la morphologie des organites cellulaires et le trafic des protéines endogènes. Au cours de cette thèse, une analyse des relations structure fonction du composé Retro-2 nous a permis d’identifier les régions de l'inhibiteur qui sont critiques pour l'activité de protection. Nous avons identifié un dérivé dihydroquinazolinone nommé Rétro-2.1 qui est à ce jour l'inhibiteur le plus puissant contre les toxines Stx. Afin d’identifier la cible moléculaire de Retro-2.1, nous avons développé des sondes photo-activables bio-actives. En outre, les données de diffraction des rayons X ont révélé que de l'activité antitoxine réside principalement dans l’énantiomère S. (S) -Retro-2.1 est 500 fois plus puissant contre Stx (50 nM) que la molécule initiale. Cette étude peut donner lieu à un nouveau concept thérapeutique ciblant la voie de transport rétrograde de la toxine à l'intérieur de la cellule hôte. Une telle stratégie thérapeutique pourrait donc être étendue à d'autres agents pathogènes qui usent également du trafic rétrograde pour une intoxication des cellules hôtes. Ce nouveau concept thérapeutique qui permet de cibler les cellules hôtes et non l'agent pathogène représente une véritable percée dans la découverte de médicaments à large spectre et réduit le risque de développement d’une résistance chez l’agent pathogèneShiga toxins (Stx) are produced by Shigella dysenteriae and certain species of E. coli that can be transmitted to humans primarily through consumption of contaminated foods and may cause severe disease. Stx is released by the bacteria in the intestine and subsequently, could cross the downstream blood vessels to reach their main target organs such as kidney. Damage to the kidney can result in serious life-threatening complication hemolytic uremic syndrome, for which there is no proven safe treatment available other than supportive care. Stx invades renal endothelial cells in a retrograde manner from cell surface to the endoplasmic reticulum in order to gain access to its cytosolic target, 28S rRNA. By using HTS, it was previously demonstrated that the compound Retro-2 blocks retrograde trafficking of Stx at the early endosome-TGN interface, without affecting the morphology of cellular organelles and trafficking of other endogenous proteins. In this work, different regions of the lead inhibitor Retro-2 that are critical for the protective activity have been determined by systematic structure-activity relationship studies. It allowed us to identify a dihydroquinazolinone derivative, named Retro-2.1 that is the most potent inhibitor of Stx to date and also to develop bio-active photo-activatable probes with the aim of identifying the molecular target of Retro-2 derivatives. Further, crystal X-ray diffraction data revealed that the antitoxin activity resides mainly in the S-enantiomer. (S)-Retro-2.1 has displayed 500 fold more potency (50 nM) than parent molecule against Stx cytotoxicity. This study may result in a new therapeutic concept - targeting the retrograde transport route of toxin inside host cell - for the treatment of Stx-producing E. coli infections and could therefore be extended to other pathogens that also traffic via the retrograde transport. Such a new therapeutic concept that target the host cells and not the pathogen itself would represent a real breakthrough in drug discovery leading to broad spectrum drugs
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Lindemann, Dirk, Ursula Berka, and Martin Volker Hamann. "Early Events in Foamy Virus - Host Interaction and Intracellular Trafficking." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-178848.
Full textAbstract:
Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.
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Sorre, Benoît. "Role of membrane curvature in intracellular trafficking : 3 case studies." Paris 7, 2010. http://www.theses.fr/2010PA077021.
Full textAbstract:
Le trafic intracellulaire est une des grandes fonctions cellulaire, consistant en un échange permanent de structures membranaires transportant des lipides et des protéines d'un compartiment de la cellule vers un autre. Un nombre croissant de preuves expérimentales indique que la courbure membranaire y joue un rôle important, que ce soit pour le tri ou pour la régulation de l'activité de certaines protéines. Pour étudier l'influence de la courbure lors de ces processus, j'ai mis au point un dispositif expérimental in vitro permettant de tirer des nanotubes de membrane de rayon contrôlé à partir de vésicules géantes unilamellaires (GUV), combiné à un microscope confocal. Ce système permet de faire varier le rayon du tube dans la gamme 10-100nm, en mesurant quantitativement l'évolution de sa composition. Nous avons d'abord utilisé ce dispositif expérimental pour montrer que sous certaines conditions la courbure de la membrane peut induire un tri de lipides. En parallèle, nous avons étudié les conséquences de l'activité courbure-dépendante d'une enzyme (ArfGAPl) sur la localisation de son substrat (Arfl) dans une système membranaire ou une région très courbée est connectée a un réservoir de membrane plane. Enfin, nous avons utilisé cette même approche pour caractériser les capacités d'une protéine a domaine N-BAR (amphiphysin 1) a déformer les membranesIntracellular trafficking thus consists in a permanent exchange of membrane structures transporting lipids and proteins from a compartment of the cell to another. T. Membrane is not only the passive envelope of transport intermediates and the physico-chemical properties of the membrane have to be taken into account. In particular, a growing number of experimental evidence shows that membrane curvature might play an important part in the regulation of intracellular processes. Indeed, transport intermediates are very curved objects (R< 50nm) compared to the membrane from which they are formed. This problem being difficult to study inside the cell, we have used an in vitro approach consisting in pulling membrane nanotubes out of Giant Unilamellar Vesicles. It allows setting the tube radius in the 10-100nm range while monitoring its composition thanks to fluorescence and force measurements. We have used this approach to show that membrane curvature is able to induce lipid sorting and under which conditions. We have also been interested in which way the curvature-dependent enzymatic activity of ArfGAPl affects the spatial localization of its substrate (Arfl) in a membrane configuration where a highly curved membrane part is connected to a fiat membrane reservoir Finally, we have been interested to the membrane deformation capabilities of amphiphysin1, a protein belonging to the BAR domain protein family
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Grandinetti, Giovanna. "Mechanisms of Cytotoxicity and Intracellular Trafficking for Gene Delivery Polymers." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/77142.
Full textAbstract:
Herein, different polymer libraries were examined to determine the effect polymer structure has on intracellular events. The effect of different polyamine lengths in copolymers on cellular uptake, the effect of modifying end groups of trehalose-containing polymers on transfection efficiency, and the effect of different linker lengths between galactose and a hepatocyte-targeted polymer on transfection efficiency were studied. Furthermore, it was demonstrated that polymers with terbium chelated in their repeat units could potentially be used for Förster resonance energy transfer (FRET) studies to monitor pDNA release from the polymer. Much of the work in this dissertation focuses on elucidating the intracellular mechanisms of linear poly(ethylenimine) (PEI) and how it compares to poly(L-tartaramidopentaethylenetetramine) (T4) and poly(galactaramidopentaethylenetetramine) (G4), two poly(glycoamidoamine)s synthesized by our group. The long-term goal of this project is to develop structure-function relationships between polymers and pDNA delivery efficacy that will result in the rational design of safe, efficient vehicles for therapeutic nucleic acid delivery.
Many polymers used as DNA delivery vehicles display high cytotoxicity. Often, the polymers with the highest transfection efficiency are the most toxic, as demonstrated herein by PEI and T4 with varying polymer lengths. Therefore, it was of interest to study how polymer structure influences mechanisms of cytotoxicity. To this end, studies on several mechanisms of cytotoxicity, including nuclear envelope permeabilization, were conducted. Longer polymers induced more cytotoxic responses than shorter ones, and it appears that hydroxyl groups in the repeat unit of polymers play a role in polyplex formation. This research has also led us to a potential link between transfection efficiency and cytotoxicity; the polymers with the highest transfection efficiency were also the most toxic, and were also able to induce the most nuclear envelope permeability. It is possible that these polymers' ability to permeabilize the nuclear envelope is what causes their high transfection efficiency and high toxicity. In addition, flow cytometry and confocal microscopy studies revealed that polymer structure plays a role in nuclear trafficking; poly(glycoamidoamine)s G4 and T4 more dependent on intracellular machinery than PEI. This research demonstrates the impact that changes in polymer structure have on intracellular mechanisms.Ph. D.
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Lindemann, Dirk, Ursula Berka, and Martin Volker Hamann. "Early Events in Foamy Virus - Host Interaction and Intracellular Trafficking." MDPI, 2013. https://tud.qucosa.de/id/qucosa%3A28912.
Full textAbstract:
Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.
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Parman, Jaime Lyn. "The Role of ERp57 in Hras Intracellular Trafficking and Function." Digital Commons @ East Tennessee State University, 2003. https://dc.etsu.edu/etd/844.
Full textAbstract:
Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led to a decrease of active ras. Second, specific anti-ERp57 antibodies were delivered into 3T3 cells expressing GFP-ras chimeras to observe the effect on intracellular trafficking. Anti-ERp57 antibodies blocked Hras plasma membrane localization but not Kras suggesting that ERp57 may be involved in Hras intracellular trafficking and function.
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Rohl, Joan. "Intracellular trafficking and secretion of matrix metalloproteinases during macrophage migration." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/102374/1/Joan_Rohl_Thesis.pdf.
Full textAbstract:
This thesis advances the knowledge of intracellular trafficking pathways for the cell surface delivery of Matrix metalloproteinases 9 and 14 in macrophages. As elevated and persistent levels of these proteolytic enzymes contribute to excessive inflammation and poor wound healing outcomes, the findings from this thesis could lead to the development of improved therapeutics for the treatment of chronic wounds. Trafficking machinery proteins responsible for cell surface delivery of Matrix metalloproteinase 14, matrix degradation and macrophage invasion was identified and could be used as novel therapeutic targets.
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West, Zoe. "Characterising the intracellular trafficking of matrix metalloproteinase 14 in macrophages." Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/213196/1/Zoe_West_Thesis.pdf.
Full textAbstract:
This thesis further elucidated the intracellular trafficking pathway of matrix metalloproteinase 14 (MMP14) in macrophages. It specifically investigated the trafficking pathway utilised by MMP14 for surface delivery in macrophages and the regulatory proteins that aid in this trafficking pathway.
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LETTIERO, BARBARA. "Uptake and intracellular trafficking of nanoparticles for potential medical use." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/20247.
Full textAbstract:
My PhD project has been focused on the characterization of the in vitro behavior of engineered Nanoparticles (NPs), especially concerning the first phases of interaction with barriers of different nature present in the body : a model of air-blood barrier , e.g. alveolar epithelial A30 cell line and an immunological barrier represented by the activation of the complement system. In light of these considerations, I have first compared the mechanism of cellular uptake and intracellular distribution of Solid Lipid NPs and Iron Oxide NPs over time. Then, I have studied the likely molecular basis of in vitro complement activation by NPs, as it can be strongly associated to acute pseudoallergic reactions in sensitive individuals within minutes of NPs infusion.
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Dharwada, Sai Tejasvi. "Intracellular trafficking of a model polytopic membrane protein in Saccharomyces cerevisiae." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52727.
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Polytopic membrane protein synthesis involves translation followed by translocation across the lipid bilayer at the ER. Here, various chaperones, together with enzymes that add post and co-translational modifications, help the protein achieve a final three-dimensional structure. General and substrate-specific chaperones prevent toxic aggregation of proteins by shielding and preventing interaction between non-native species. Terminally misfolded proteins are destroyed by the quality control machinery of the cell and the amino acids are recycled for further use.
In the following study, we used Chitin synthase III (Chs3) of Saccharomyces cerevisiae as a model to dissect the complexities involved in polytopic membrane protein synthesis at the ER. Previous genetic screens from our lab have revealed a novel regulator of Chs3 trafficking called Pfa4, a DHHC enzyme required for Chs3 palmitoylation at the ER. At the ER, Chs3 also requires Chs7, a dedicated chaperone for folding and assembly. We identified a novel secondary role for Chs7 in Chs3 trafficking as a co-factor required for Chs3 function at the plasma membrane. Our study also examined the role of palmitoylation in Chs3 trafficking. Palmitoylation of Chs3 is required for its efficient interaction with Chs7, in addition to folding and ER export. A genome-wide screen also identified the Ubp3/Bre5 deubiquitination complex as a regulator of non-lipidated Chs3 degradation at the ER. The discovery that dedicated chaperones can take on additional roles and that palmitoylation can influence chaperone-client interactions could provide insights into the workings of the protein folding machinery at the ER.Medicine, Faculty of
Graduate
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Agnihothram, Sudhakar Srinivasarao. "Biogenesis, Assembly and Intracellular Trafficking of Junin Arenavirus Envelope Glycoprotein Complex." The University of Montana, 2009. http://etd.lib.umt.edu/theses/available/etd-05192009-095740/.
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An unusual feature in the arenavirus envelope glycoprotein complex (GP-C) is the presence of a myristoylated stable signal peptide (SSP) in addition to the receptor binding subunit G1 and the transmembrane fusion subunit G2. Genetic studies were employed to understand the structure-function of the GP-C complex, with emphasis on elucidating the role of SSP in arenavirus life cycle. We present genetic evidence that support the inclusion of G2 as a member of Class I viral fusion protein, where membrane fusion is mediated by six-helix bundle structure as in HIV and Influenza viruses. Furthermore, we have identified crucial roles for SSP in the GP-C complex. In the mature glycoprotein complex, SSP assumes a bitopic membrane topology with both its N and C termini in the cytosol, and a short ectodomain loop. This membrane orientation allows it to mask the endogenous endoplasmic reticulum retrieval signals in the cytoplasmic tail of G2 thereby allowing the transit of fully assembled GP-C complex through the Golgi to the cell surface. SSP also interacts with the ectodomain of G2 on the outer surface of the membrane. This interaction is critical in modulating the pH at which the membrane fusion is activated. The SSP-G2 pocket has been identified as the target of newly discovered small-molecule inhibitors of arenaviral entry. Beyond its role in intracellular trafficking and pH-dependent membrane fusion of GP-C complex, SSP might be involved in virus assembly and budding. Studies employing immunogold electron microscopy indicated that GP-C complex clusters into microdomains of 120 nm size independent of other viral proteins. Clustering of GP-C into membrane microdomains is neither influenced by SSP myristoylation nor by the co-expression of the matrix protein do Z. Regions of plasma membrane containing Z not co-localize with GP-C containing microdomains. Clustering of proteins or lipids on the plasma membrane may bring Z and GP-C together at the virus budding sites. Taken together, these data have contributed to the understanding of the unique subunit organization in GP-C complex and the mechanisms underlying efficient co-ordination of these subunits to execute significant functions in the arenavirus life cycle.
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Kern, Beate. "Analysis of Helicobacter pylori VacA-containing vacuoles and VacA intracellular trafficking." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-184058.
Full textAbstract:
The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases like gastritis, duodenal and gastric ulcers, and gastric cancer. It has evolved a range of mechanisms to aid in colonization and persistence, manipulating the host immune response to avoid clearance. A major factor in this is the secreted vacuolating cytotoxin VacA which has a variety of effects on host cells. VacA is endocytosed and forms anion-selective channels in the endosome membrane, causing the compartment to swell. The resulting VacA-containing vacuoles (VCVs) can take up most of the cellular cytoplasm. Even though vacuolation is VacA's most prominent and namesake effect, the purpose of the vacuoles is still unknown.
VacA exerts influence on the host immune response in various ways, both pro- and anti- inflammatorily. Most importantly, it disrupts calcium signaling in T-lymphocytes, inhibiting T-cell activation and proliferation and thereby suppressing the host immune response. Furthermore, VacA is transported to mitochondria, where it activates the mitochondrial apoptosis pathway. Within the cell, VacA has only been shown to localize to endocytic compartments/VCVs and mitochondria. Considering its diverse effects, however, the existence of other cellular sites of action seems plausible.
In this study, the VCV proteome was comprehensively analyzed for the first time in order to investigate VCV function. To this end, three different strategies for VCV purification from T-cells were devised and tested. Eventually, VCVs were successfully isolated via immunomagnetic separation, using a VacA-specific primary antibody and a secondary antibody coupled to magnetic beads. The purified vacuoles were then measured by mass spectrometry, revealing not only proteins of the endocytic system, but also proteins usually localized in other cellular compartments. This apparent recruitment of proteins involved in all kinds of cellular pathways indicates a central function of VCVs in VacA intoxication effects.
In a global evaluation, the VCV proteome exhibited an enrichment of proteins implicated in immune response, cell death, and cellular signaling; all of these are processes that VacA is known to influence. One of the individual proteins contained in the sample was STIM1, a calcium sensor normally residing in the endoplasmic reticulum (ER) that is important in store- operated calcium entry (SOCE). This corroborates the findings of a concurrent report, in which VacA severely influenced SOCE and colocalized with STIM1. A direct interaction of STIM1 with VacA was examined in a pull-down assay, but could be neither shown nor excluded.
Immunofluorescence experiments conducted in HeLa cells confirmed the presence of VacA in the ER and also found it to traffic to the Golgi apparatus, identifying these two cellular compartments as novel VacA target structures. The exact route of VacA transport remains unclear, but the involvement of both the ER and the Golgi suggests the possibility of retrograde trafficking, analogous to other bacterial toxins like shiga and cholera toxins.
In summary, the elucidation of the VCV proteome and the discovery of the ER and the Golgi apparatus as VacA target structures have generated intriguing starting points for future studies. The detection of many proteins implicated in VacA intoxication effects in the VCV proteome leads to the proposal of VCVs as signaling hubs that may coordinate the complex meshwork of VacA effects. Further investigation of individual proteins is expected to help greatly in illuminating this matter.
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Hofmann, Daniel [Verfasser]. "Drug delivery, entry and intracellular trafficking of polymeric nanoparticles / Daniel Hofmann." Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/1062824466/34.
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Bowness, Peter William. "Intracellular zinc and copper : sequestration and trafficking in Anabaena PCC 7120." Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413939.
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Coppola, Stefano, Daniela Pozzi, Giulio Caracciolo, and Thomas Schmidt. "Intracellular trafficking of lipoplexes: a particle image correlation spectroscopy (PICS) study." Diffusion fundamentals 20 (2013) 27, S. 1, 2013. https://ul.qucosa.de/id/qucosa%3A13592.
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Slowing, Igor Iván. "Mesoporous silica nanoparticles as vehicles for intracellular trafficking and controlled release." [Ames, Iowa : Iowa State University], 2008.
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Das, Lipsa, and Lipsa Das. "Regulation of Intracellular Trafficking of Laminin Binding Integrins in Prostate Cancer." Diss., The University of Arizona, 2017. http://hdl.handle.net/10150/625651.
Full textAbstract:
Laminin binding integrins (α6β1 and α3β1) are persistently but differentially expressed throughout prostate cancer progression and metastasis. Prostate cancer primarily invades through laminin rich nerve for extracapsular escape during cancer metastasis. An intense expression of the pro-metastatic α6 integrin was observed during perineural invasion with a heterogeneous distribution of the integrin on the cancer cell membrane as well as intracellularly. Bone and soft tissue metastasis of human prostate cancer demonstrated a similar pattern where 75-80% of the cancers had significant intracellular staining. This was correlated with an mRNA overexpression of various intracellular trafficking regulators. Using a prostate cancer cell culture model of DU145 cells, the α6 integrin was found to be constitutively internalized in cancer cells at a rate of 3.25 min-1, which was 3 fold greater than internalization rate of α3 integrin, classically considered a "non-circulating" receptor.
α6 and α3 integrins function coordinately to regulate cell migration during development, wound healing. Their orchestrated redistribution during these processes is well-known, but the mechanism remains elusive. Current study identifies intracellular trafficking of these integrins as a key mechanism of their coordination. Depletion of α3 integrin in prostate cancer cells significantly increased internalization of α6 integrin up to 1.7-fold and increased localization of α6 integrin at cell-cell membrane locations. There was a concomitant 1.8-fold increase in cell migration significantly dependent on α6 integrin. Depletion of α6 integrin expression however, had no effects on the internalization of α3 integrin indicating that the identified coordination was unidirectional.
α6 integrin trafficking drives cancer invasion, but its selective regulators are unknown. Here, Rab11FIP5 was identified as a selective regulator of α6 integrin recycling to cell membrane. Interestingly, α6 integrin was found to be primarily recycled to the cell-cell membranes where it colocalized with Rab11 and Rab11FIP5. Depletion of Rab11FIP5 reduced such membrane expression of α6 integrin, inhibited cell-cell cohesion in 3D culture and significantly reduced cell migration. The localization of α6 and α3 integrin at these locations have been implicated in cell adhesion. Based on current study α6 recycling by Rab11FIP5 might be key to such function.
Another Rab11 effector protein Rab11FIP1 was identified as a regulator of both α3 and α6 integrin trafficking. Depletion of Rab11FIP1 reduced membrane expression of α3 integrin by significantly increasing its internalization and reducing the recycling. There was a major effect on α6 integrin internalization, which increased to an extent similar to that observed on α3 integrin depletion. Rab11FIP1 regulated α6 integrin recycling, in a pathway found to be independent of Rab11FIP5.
Taken together, current research defined Rab11FIPs as regulators of α6 and α3 integrins. A unidirectional coordination between α6 and α3 integrin was identified such that loss of α3 integrin, representative of high grade prostate cancer, amplifies integrin α6 integrin internalization and a resultant migratory phenotype.
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Llinares, Elisa. "Function, regulation and intracellular trafficking of the vacuolaryeast pq-loop (Ypq) proteins." Doctoral thesis, Universite Libre de Bruxelles, 2012. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209704.
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The cytoplasm of eukaryotic cells contains several membrane-delimited compartments of specific molecular compositions and functions. Among those, the vacuole of fungal cells is often described as an organelle equivalent to the lysosomes of animal cells and the vacuoles of plant cells. These compartments indeed share two similar features: they contain a wide variety of hydrolases and are the most acidic compartments of the cell, which accounts for their key role in the intracellular degradation of macromolecules. In humans, dysfunctions of the lysosomes often give rise to lysosomal related diseases, such as lysosomal storage disorders. These are a class of metabolic disorders caused by the accumulation of non-degraded macromolecules or impaired export of hydrolytic degradation products. Cystinosis is an autosomal recessive disorder (1/200 000 incidence) generally associated with renal dysfunctions. It is caused by the accumulation and crystallization of cystine, the disulfide of cysteine, into the lumen of lysosomes. Cystinosin, the causative gene product of cystinosis, is present at the lysosomal membrane and catalyses the export of cystine from this compartment. The human cystinosin is a member of the Lysosomal Cystine Transporter (LCT) family. LCT proteins are conserved in all eukaryotic species and are defined by the presence of highly conserved PQ-loop motifs. During this thesis work, we have studied three LCT proteins of the yeast Saccharomyces cerevisiae, named Ypq1, Ypq2 and Ypq3 (Yeast PQ-loop proteins 1, 2 and 3). We first showed that these proteins localize to the vacuolar membrane. We next studied the roles of these proteins, the regulation of their genes and the mechanisms and signals implicated in their delivery to the vacuolar membrane. We also contributed to the functional characterization of a mammalian homologue of yeast Ypq proteins, named rPqlc2.
In the first part of this work, we report that the Ypq proteins are most probably implicated in the export of basic amino acids from the vacuole to the cytosol. More precisely, Ypq2 and Ypq3 behave like vacuolar arginine and lysine exporters, respectively. Interestingly, the mammalian rPqlc2 protein expressed in yeast reaches the vacuolar membrane and functions as an orthologue of the Ypq proteins. Our results also reveal that the expression of the YPQ3 gene is regulated by the Lys14 transcription factor, responsible for the transcriptional activation of the LYS genes encoding enzymes implicated in the biosynthesis of lysine. We have also noted that, in general, the expression of the expression of the YPQ genes is regulated according to the quality of the nitrogen source available in the extracellular medium, eg. YPQ3 is sensitive to the nitrogen catabolite repression regulatory mechanism.
In the last part of this thesis work, we investigated the intracellular trafficking of the Ypq proteins and show that these predominantly reach the vacuolar membrane via the ALP (alkaline phosphatase) pathway due to the presence of a dileucine-based sorting signal in their sequences. Interestingly, a similar mechanism seems responsible for targeting to the yeast vacuole of the mammalian rPqlc2 protein.
Une caractéristique des cellules eucaryotes est leur organisation en compartiment internes délimité par une membrane lipidique, appelé organelles. Ces compartiments intracellulaires présentent une composition lipidique et protéique particulaire conforme à leur identité et fonction. Les lysosomes de cellules de mammifères et la vacuole fongique jouent un rôle clé dans la digestion intracellulaire de macromolécules et de ce fait leurs lumières sont enrichis d’enzymes hydrolytiques nécessaires à cette action. Des disfonctionnements du lysosome peuvent être la conséquence de pathologie chez l’homme, regroupé sous le nom de maladie lysosomale, lié à un à une accumulation de macromolécules non digéré ou un default d’export des produits d’hydrolysé depuis la lumière du lysosome. La cystinose est une maladie autosomale récessive avec une faible fréquence d’incidence (1/200 000) qui regroupe trois formes cliniques :deux formes rénales graves et une forme extra-rénale. Cette maladie est due à une accumulation et cristallisation de cystine dans la lumière du lysosome qui est corrélé à des mutations ponctuelles dans le gène CTNS qui code pour l’exporteur de cystine, la cystinosine. Cette protéine est un membre de la famille LCT (Lysosomal Cystine Transporter) qui possède des représentants chez les cellules animales, végétales et fongiques. Les protéines de la famille possèdent une taille et une topologie prédite similaire (7 segments transmembranaires) et on retrouve aussi au sein de ces protéines deux exemplaires de motifs PQ. Lors de ce travail de thèse nous nous sommes intéressés à trois membres de la famille LCT chez Saccharomyces cerevisiae que nous avons nommé Ypq1, Ypq2 et Ypq3 pour Yeast PQ-loop proteins. Ces protéines n’ayant pas fait l’objet de nombreuses études, nous nous sommes orientés vers une analyse fonctionnelle et transcriptionnelle. De plus, nous avons également étudié les mécanismes et signaux impliqué dans leur adressage vers la vacuole. Finalement, nous avons également inclus dans notre étude un homologue mammalien de ces protéines, rPqlc2.
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Doctorat en Sciences
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Lux, Kerstin. "Visualization of the intracellular trafficking of incoming Adeno-associated virus type 2." Diss., kostenfrei, 2006. http://edoc.ub.uni-muenchen.de/8690/.
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Remelli, Rosaria. "Trafficking and intracellular interactions of Group I mGluRs and their splice variants." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433330.
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Dillon, Christian. "Intracellular trafficking, and function of receptor tyrosine kinases in mammary gland development." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397232.
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Kaur, Satdip. "Role of the CTLA-4 C-terminus in regulating its intracellular trafficking." Thesis, University of Birmingham, 2014. http://etheses.bham.ac.uk//id/eprint/4903/.
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CTLA-4 is an important inhibitor of T cell immune responses. The location of CTLA-4 in intracellular vesicles is the most dominating aspect of its biology, yet the significance of this at the functional level remains to be completely understood. I have therefore investigated the role of the CTLA-4 cytoplasmic domain in the intracellular trafficking of the receptor with particular emphasis on sorting signals encoded within this domain. We found that CTLA-4 was located in punctate intracellular vesicles in transfected cells, activated T cells and in regulatory T cells. CTLA-4 internalisation from the cell surface was clathrin dependent and was driven by the YVKM motif encoded within the cytoplasmic domain. Post-internalisation CTLA-4 colocalised with markers of late endosomes. Since the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression we investigated this further and found that ubiquitination of intracellular lysine residues targets CTLA-4 to lysosomes. The ability of CTLA-4 to recycle was dependent on the YVKM motif and subtle changes in this motif reduced recycling efficiency. Moreover, in the absence of lysine residues CTLA-4 recycling was enhanced. CTLA-4 transendocytosis was conserved through evolution but the exact sorting signals required for this function remain to be identified. Overall this thesis emphasises the importance of the CTLA-4 cytoplasmic domain in regulating its intracellular trafficking.
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Azuma, Yuya. "Studies on intracellular trafficking and degradation of ABCA1 involved in HDL formation." Kyoto University, 2009. http://hdl.handle.net/2433/126527.
Full textAbstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第14841号
農博第1781号
新制||農||974(附属図書館)
学位論文||H21||N4481(農学部図書室)
27247
UT51-2009-F483
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 和光, 教授 阪井 康能, 教授 三芳 秀人
学位規則第4条第1項該当
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Irandoust, M. "Intracellular trafficking of G-SCSF receptor; mechanisms and implications of signaling function." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/13950.
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Lauwers, Elsa. "Role of sphingolipids and polyubiquitin chains in intracellular trafficking of the yeast GAP1 permease." Doctoral thesis, Universite Libre de Bruxelles, 2007. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210648.
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In the past fifteen years, ubiquitin has emerged as a central regulator of membrane protein trafficking. In this context, covalent attachment of this small protein to lysine residues of cargo proteins, a reversible modification termed ubiquitylation, provides a signal for their targeting to the vacuolar/lysosomal lumen where they are degraded, both in yeast and higher eukaryotes. Ubiquitylation is also used as a means of controlling the function of specific proteins in several trafficking machineries. The role of lipids - and in particular of membrane domains named lipid rafts - in controlling the intracellular trafficking of membrane proteins has also been the subject of intense investigation in recent years.One of the membrane proteins of the yeast Saccharomyces cerevisiae whose intracellular trafficking has been extensively studied is the general amino acid permease Gap1. Yet some aspects of the function of ubiquitin in the nitrogen-dependent control of this protein remain controversial. Moreover, the potential role of lipid rafts in regulating the functional properties and traffic of the Gap1 permease had not been investigated before this thesis work.
The first part of our work readdresses the role of Gap1 ubiquitylation, and more precisely of the modification of the permease with polyubiquitin chains linked through the lysine 63 of ubiquitin, in controlling the fate of this protein in the secretory pathway. Our observations indicate that nitrogen-induced ubiquitylation of newly synthesised Gap1 occurs in the trans-Golgi complex. However, contrary to the generally accepted view, this modification is not necessary for the permease to exit this compartment en route to the endosome but only for its subsequent targeting to the vacuolar lumen via the multivesicular body (MVB) pathway. Our results also provide evidence that K63-linked polyubiquitylation is important mostly at the late endosomal level, for proper sorting of Gap1 into the MVB pathway, whether the permease comes from the cell surface by endocytosis or directly from the secretory pathway.
In the second part of this work, we present a set of data providing novel insights into the controversial question of the exact nature of lipid rafts in yeast. We first showed that the Gap1 permease is associated with detergent-resistant membranes (DRMs) - the proposed biochemical equivalent of lipid rafts - when it is located at the cell surface. Our data further suggest that this may be true for most if not all yeast plasma membrane proteins. Moreover, we found that Gap1 production must be coupled to de novo synthesis of sphingolipids (SLs), major constituents of rafts, in order for the newly synthesised permease to be correctly folded, active, associated with DRMs, and stable at the cell surface. We propose a model where Gap1 would associate with newly synthesised SLs during its biogenesis and/or secretion, this association shaping the permease into its native conformation and ensuring its incorporation and stabilisation in specific lipid domains at the plasma membrane. Failure of Gap1 to acquire this lipidic microenvironment in turns leads to its ubiquitin-dependent degradation by a quality-control mechanism. This model might be valid for many other plasma membrane proteins and might account for their lateral distribution between distinct membrane domains.
Doctorat en Sciences
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Allaire, Patrick. "Biochemical and functional characterization of connecdenn and its DENN domain in intracellular trafficking." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66644.
Full textAbstract:
Endocytosis and vesicle trafficking are essential for cellular division, differentiation and survival. Following endocytosis, cargo is delivered to the sorting endosome, where it is selected for degradation or recycled back to the cell surface. The various trafficking steps within the endosomal system are regulated by Rabs, small GTPases, which toggle between an inactive GDP-loaded conformation and an active GTP-loaded conformation. Clathrin-mediated endocytosis (CME) allows cargo internalization and coupling to the endosomal system via formation of clathrin-coated vesicles (CCVs). Connecdenn was identified as a component of clathrin-coated vesicles (CCVs) isolated from developing rat brain. The C-terminus of connecdenn is predicted to be unstructured and encodes several binding motifs that bind to the CME proteins intersectin1, endophilinA1 and AP-2. Connecdenn is enriched in brain, localizes to synapses, and its knockdown in hippocampal cultures inhibits synaptic vesicle recycling. Connecdenn enriches strongly on CCVs and is stably associated with the CCV membrane after removal of its clathrin coat. The stable membrane association of connecdenn depends on the N-terminal DENN domain, an evolutionary conserved but uncharacterized tri-partite domain. The DENN domain also functions as a guanine nucleotide exchange factor (GEF) for Rab35, which regulates cargo recycling. In fact, in non-neuronal cells, connecdenn and Rab35 do not regulate CME but control the recycling of cargo from sorting endosomes. Curiously, connecdenn/Rab35 specifically control the recycling of MHCI, which enters the cell independently of CME but do not influence the trafficking of the transferrin receptor, a prototypical CME cargo. Overall, my thesis work on connecdenn has allowed us to appreciate three important conclusions. First, connecdenn is a CCV protein that functions at the neuronal synapse to regenerate SVs. Second, connecdennLe trafic intracellulaire est un processus essentiel pour la division, différenciation et survie cellulaire. Suite à l'endocytose, le cargo est acheminé vers l'endosome de triage qui le dirigera vers la voie de dégradation ou le recyclera à la surface de la cellule. Le trafic endosomal est régi par les petites GTPases Rab, basculant entre la conformation inactive GDP-chargé et active GTP-chargé. Dans l'endocytose tributaire pa r la clathrine (ETC), la formation de vésicules tapissées de clathrine (VTC) permet l'internalisation du cargo et son couplage au système endosomal. La protéine Connecdenn a été identifiée comme composante des VTCs. L'extrémité C-terminale de Connecdenn est prédite etre non structurée et code plusieurs motifs d'interaction avec les protéines impliquées dans l'ETC comme intersectin1, endophilinA et AP-2. Connecdenn est enrichie sur les VTCs. Lorsque le manteau de clathrine est dissocié, l'association de Connecdenn avec la membrane des VTC demeure stable. Cette association et son recrutement dans les VTCs s'effectuent grâce à son extrémité C-terminale et s'avèrent nécessaires pour le recyclage des vésicules synaptiques. Par contre, l'association de Connecdenn avec la membrane des VTCs dépend de son domaine DENN N-terminal, dont la fonction n'a pas encore été caractérisée. Ce domaine fonctionne également comme un facteur d'échange guanine (FEG). En fait, dans les cellules non-neuronales, Connecdenn et Rab35 ne régularisent pas l'ETC, mais plutôt le recyclage du cargo à partir des endosomes de triage. De plus, l'épuisement de Connecdenn ou Rab35 perturbe spécifiquement le recyclage de MHCI, dont l'internalisation n'est pas régie par l'ETC. Toutefois, l'absence de Connecdenn ou de Rab35 n'a aucune influence sur le trafic du récepteur de la transferrine, cargo typique de l'ETC. Mon travail sur Connecdenn se résume a trois conclusions generales;
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Danquah, Kwabena Owusu. "Intracellular trafficking of baculovirus in insect cells and its application in gene therapy." Thesis, Oxford Brookes University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543801.
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Guet, David. "Architecture of the Golgi apparatus and membrane trafficking probed by intracellular optical micromanipulation." Paris 6, 2012. http://www.theses.fr/2012PA066203.
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Le trafic membranaire est basé sur la formation d'intermédiaires de transports qui transitent d'un compartiment à un autre. Des études in vitro ont montré que des paramètres physiques tels que la courbure, la tensione et la composition des membranes influence le bourgeonnement et la fission de ces intermédiaires. Plus récemment le rôle du cytosquelette d'actine dans la fission de ces vésicules a été mis en avant. Au cours de mon doctorat je me suis intéresé à l'effet d'un stress mécanique sur la formation de vésicules in cellulo. Dans cette thèse je montre que l'utilisation de la microscopie confocale, couplée à une pince optique, permet de visualiser en temps réel des déformations induites par la sonde sur des membranes de l'appareil de Golgi exprimant des protéines GFP et de mesurer les forces nécessaire à cette déformation. Mes résultats montrent que la contrainte mécanique induit une déformation à longue portée, suggérant l'existence d'une matrice englobant l'appareil de Golgi. La dépolymérisation de l'actine, candidate pour cette matrice, entraîne une diminution des forces nécessaires aux déformations. Enfin, l'application de la contrainte mécanique entrapine un défaut de la formation de vésicules positives pour Rab6 et la formation de longues structures tubulairesMembrane transport is based on the formation of tubulo-vesicular intermediates traveling from one compartment of the cell to another along cytoskeletal tracks. In vitro studies have shown that physical parameters, such as membrane curvature, tension and composition, influence the budding and fission of transport intermediates. Recent studies in cells have highlighted the central role of the actin cytoskeleton in the fission of transport intermediates from the Golgi apparatus. Here I investigate the role of a mechanical stress on intracellular transport in cellulo. I focus on the mechanics of Golgi membranes and the formation of transport intermediates from the Golgi apparatus. Using confocal microscopy, I visualize the deformation of Rab6-positive and COPI-positive Golgi membranes applied by an internalized microsphere trapped in an optical tweezers, and simultaneously measure the corresponding forces. My results show that the force necessary to deform Golgi membranes drops when the actin cytoskeleton is depolymerized, suggesting that actin strongly contributes to the local rigidity of the Golgi apparatus. We also show that the applied stress has a long-range effect on Golgi membranes and induces a sharp decrease in the formation of vesicles and the formation of tubular structures from the Golgi apparatus
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Stamey, Jennifer Anne. "Systemic and Intracellular Trafficking of Long-chain Fatty Acids in Lactating Dairy Cattle." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/38689.
Full textAbstract:
Marine oils are used as ration additives to provide omega-3 fatty acids to dairy
cows. Supplementing dairy cows with omega-3 fatty acid-rich feeds does not easily
increase quantities in milk fat of dairy cows because polyunsaturated fatty acids are
biohydrogenated in the rumen. Lipid encapsulation of omega-3 fatty acids provides
protection from biohydrogenation in the rumen and allows them to be available for
absorption and utilization in the small intestine. Lactating cows were supplemented with
rumen protected algae biomass or algal oil in a 4 Ã 4 Latin Square. Feeding lipid
encapsulated algae supplements increased docosahexaenoic acid content in milk fat while
not adversely impacting milk fat yield; however, docosahexaenoic acid was preferentially
esterified into plasma phospholipid, limiting its incorporation into milk fat. In the second
study, triglyceride emulsions of oils enriched in either oleic, linoleic, linolenic, or
docosahexaenoic acids were intravenously infused to avoid confounding effects of
triglyceride esterification patterns in the small intestine and to compare mammary uptake.
Milk transfer of fatty acids delivered as intravenous triglyceride emulsions was reduced
with increased chain length and unsaturation. Increased target fatty acids were evident in
plasma phospholipid, suggesting re-esterification in the liver. Transfer efficiencies were 37.8, 27.6, and 10.9±5.4% for linoleic, linolenic, and docosahexaenoic acid. Both liver
and mammary mechanisms may regulate transfer of long-chain polyunsaturates.
Intracellular fatty acid binding proteins (FABP) are cytoplasmic proteins that are
hypothesized to be essential for fatty acid transport and metabolism by accelerating longchain
fatty acid uptake and targeting to intracellular organelles, such as the endoplasmic
reticulum for triglyceride esterification. FABP3 mRNA is highly expressed in bovine
mammary and heart tissue, but is not present in MAC-T cells, a bovine mammary
epithelial cell line. When overexpressed in MAC-T cells, FABP3 does not appear to be
rate-limiting for fatty acid uptake in vitro and did not alter lipid metabolism. The function
of FABP3 in the mammary gland remains unclear.Ph. D.
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Starr, Tregei Nicole. "Quantitative Studies of Intracellular Trafficking of Two Classes of Resident Golgi Apparatus Proteins." Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/27217.
Full textAbstract:
The research presented in this dissertation consists of two primary parts. The initial focus centered on understanding the distribution of Golgi resident glycosyltransferases between the ER and Golgi at steady-state. Retrograde trafficking of these Golgi proteins has been demonstrated experimentally mandating the existence of a dynamic equilibrium between the Golgi apparatus and ER. Our published studies also included the development of a quantitative method for analysis of data collected using fluorescent microscopy. The second part of this dissertation presents results pertaining to the quantification of a unique Golgi resident protein that cycles in the late endosome bypass pathway. Using the published method of analysis and techniques developed during the initial project, the anterograde and retrograde transport kinetics of this Golgi protein were determined and used to develop a compartmental model for pH sensitive trafficking in the bypass pathway. The spatial Golgi distribution of the protein during retrograde transport to the Golgi following endosomal exit was also investigated. This research lies at the interface of experimental cell biology and quantitative computational analysis. These experiments combined more traditional experimental biological approaches with more recent computational approaches to understanding cellular mechanisms. Additionally, development of a quantitative method of analysis validated the use of fluorescent microscopy as a quantitative tool for studying intracellular proteins.Ph. D.
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Lackey, Chantal A. "pH-responsive polymer-protein complexes for control of intracellular trafficking of biomolecular therapeutics /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/8066.
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