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Jamal, Layal. "Structural and functional characterization of the lysosomal amino acid transporter PQLC2." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL129.
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PQLC2, qui signifie protéine conte- nant des répétitions de boucle proline-gluta- mine 2, appartient à une famille de protéines de transport membranaires caractérisées par une topologie membranaire à sept hélices et deux motifs proline-glutamine. PQLC2 est localisé dans la membrane lysosomale des cellules mammifères, et des études utilisant du PQLC2 recombinant exprimé dans des ovocytes de Xe- nopus ont démontré que PQLC2 est un unipor- teur qui transporte spécifiquement des acides aminés cationiques. Cependant, sa structure atomique en 3D n’a pas encore été déterminée. En plus de son rôle de transporteur, PQLC2 est également un récepteur membranaire. Lorsque la cellule est privée d’acides aminés cationiques, PQLC2 recrute à la surface du lysosome un com- plexe de trois protéines (appelé CSW) : les pro- téines GTPas-activatrices C9ORF72 et SMCR8, et WDR41, l'ancrage entre CSW et PQLC2. Le com- plexe CSW est important pour le bon fonction- nement des lysosomes. De plus, des mutations congénitales dans le gène codant pour C9ORF72 sont directement associées à deux maladies neurodégénératives. Des essais de pull-down dans des extraits cellu- laires indiquent que l’interaction d’un court mo- tif peptidique de 10 acides aminés provenant d’une boucle saillante de WDR41 (boucle WDR41-7CD) avec PQLC2 est suffisante pour le recrutement lysosomique de CSW. Afin de ca- ractériser cette interaction ainsi que le rôle fonc- tionnel de PQLC2, nous avons exprimé PQLC2 de mammifère dans la levure Saccharomyces cerevisiae et établi un protocole de purification de PQLC2 basé sur la reconnaissance entre des nanocorps anti-GFP et GFP fusionné à PQLC2. Pour améliorer la stabilité de PQLC2 purifié par détergent, nous avons introduit des mutations spécifiques le long de la séquence de la pro- téine en utilisant une approche de mutagenèse basée sur un consensus. La microscopie électro- nique à contraste négatif de PQLC2 purifié par détergent suggère que ce transporteur s’as- semble sous forme d’homotrimère, comme les autres membres de la même famille de trans- porteurs à boucle PQ. Enfin, par spectroscopie de résonance paramagnétique électronique (RPE), nous avons évalué l’interaction directe entre PQLC2 et un peptide codant la boucle WDR41. Ces expériences ont révélé le rôle de certains résidus de la boucle WDR41 dans l’in- teraction PQLC2/boucle WDR41-7CD, ainsi que l’effet d’un substrat de PQLC2PQLC2, which stands for proline-glu- tamine loop repeat-containing protein 2, be- longs to a family of membrane transport pro- teins characterized by a seven-helix membrane topology and two proline-glutamine motifs. PQLC2 is localized in the lysosomal membrane of mammalian cells, and studies using recombi- nant PQLC2 expressed in Xenopus oocytes have demonstrated that PQLC2 is an uniporter that specifically transports cationic amino acids. However, its 3D atomic structure has not yet been determined. In addition to being a trans- porter, PQLC2 is also a membrane receptor. When the cell is deprived of cationic amino acids, PQLC2 recruits at the lysosome surface a complex of three proteins (called CSW): the GTPase-activating proteins C9ORF72 and SMCR8, and WDR41, the anchor between CSW and PQLC2. The CSW complex is important for normal lysosome function. In addition, congeni- tal mutations in the gene encoding C9ORF72 are directly associated with two neurodegene- rative diseases. Pull-down assays in cell extracts indicate that the interaction of a short 10 amino acid peptide motif from a protruding loop of WDR41 (WDR41-7CD loop) with PQLC2 is sufficient for lysosomal recruitment of CSW. To characterize this interaction as well as the functional role of PQLC2, we expressed mammalian PQLC2 in the yeast Saccharomyces cerevisiae, and established a purification protocol of PQLC2 based on the recognition between anti-GFP nanobodies and GFP fused to PQLC2. To improve the stability of detergent-purified PQLC2, we introduced speci- fic mutations along the protein sequence using a consensus-based mutagenesis approach. Ne- gative-staining electron microscopy of deter- gent-purified PQLC2 suggests that this trans- porter assembles as a homotrimer, like other members of the same PQ-loop family of trans- porters. Finally, by electron paramagnetic re- sonance (EPR) spectroscopy, we assessed the direct interaction between PQLC2 and a peptide encoding the WDR41 loop. These experiments revealed the role of certain WDR41 loop resi- dues in the PQLC2/WDR41-7CD loop interac- tion, as well as the effect of a PQLC2 substrate
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SAMARANI, MAURA. "CELL DAMAGE INDUCED BY LYSOSOMAL IMPAIRMENT: STUDY OF THE ROLE OF PLASMA MEMBRANE SPHINGOLIPIDS." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/482301.
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Lysosomes are the principal site of the catabolism of sphingolipids, a class of bioactive lipids mainly associated with the external leaflet of cell plasma membrane. Several lines of evidence support a direct correlation between modifications in sphingolipid pattern and the activation of specific signaling pathways, including apoptosis and autophagy. Loss-of-function mutations in genes coding for lysosomal enzymes involved in sphingolipid catabolism result in severe clinical manifestations called sphingolipidoses. These pathologies belong to the group of Lysosomal Storage Diseases and are characterized by the accumulation of undegraded materials leading to lysosomal impairment and consequent cell damage. Until now, the molecular mechanisms by which the perturbation of lysosomal homeostasis affects cell functionality and viability are unknown.
To investigate this issue, I used an artificial in vitro model of lysosomal impairment obtained by loading human fibroblasts with 88 mM sucrose for 14 days. In these experimental conditions, the absence of invertase induces sucrose accumulation into lysosomes. I found that sucrose loaded fibroblasts are characterized by a growth slowdown and by the activation of both apoptosis and autophagy. By RNA-sequencing, approximately a thousand of genes were found to be dysregulated after sucrose loading. In particular, 56 cell cycle-related genes are downregulated, whereas 37 lysosomal-related genes are upregulated. Using biochemical approaches, I found that sucrose loading activates lysosomal biogenesis although sucrose storage inhibits lysosomal functionality. In particular, in sucrose loaded cells lipid catabolism is blocked and complex lipids, such as phospholipids, cholesterol, glycosphingolipids, and gangliosides are accumulated. Moreover, I found that sucrose loading induces the nuclear translocation of the Transcription Factor EB (TFEB), a master-gene regulator of lysosomal function, which in turn promotes the increased fusion between lysosomes and the plasma membrane. This last event leads to higher levels of sphingolipid hydrolases at the cell surface resulting in the alteration of the plasma membrane sphingolipid composition and the consequent ectopic production of pro-apoptotic and pro-autophagic ceramide. Interestingly, in sucrose loaded fibroblasts the blocking of glycosphingolipid hydrolysis at the plasma membrane results in a reduction of autophagy and apoptosis.
Similar results were also obtained in response to sphingomyelin accumulation in Niemann-Pick Type A disease (NPA). NPA is a sphingolipidosis caused by acid sphingomyelinase deficiency which leads to sphingomyelin storage. Interestingly, using NPA-derived human fibroblasts loaded with 50 µM exogenous sphingomyelin for 30 days, I found that the lysosomal impairment caused by sphingomyelin accumulation activates the same molecular pathways described in healthy fibroblasts subjected to sucrose loading.
A pathogenic role of TFEB has also been suggested by biochemical analysis on brains from Acid Sphingomyelinase Knockout (ASMKO) mice. In fact, ASMKO mouse brains are characterized by TFEB nuclear translocation, increased lysosomal biogenesis, increased glycohydrolytic activities and onset of apoptosis and autophagy.
Collectively, these data suggest the existence of a cross-talk among lysosomes and the cell plasma membrane. In this context, the lysosomal impairment caused by the accumulation of uncatabolized substrates leads to an altered composition of plasma membrane sphingolipids resulting in the ectopic production of ceramide which in turn is responsible for the onset of cell damage.
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Schröder, Bernd. "Proteomanalyse der humanen lysosomalen Membran /." Marburg : Görich & Weiershäuser, 2007. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=016450683&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Johansson, Ann-Charlotte. "Lysosomal membrane permeabilization : a cellular suicide strategy /." Linköping : Department of Clinical and Experimental Medicine, Linköping University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-11614.
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Johansson, Ann-Charlotte. "Lysosomal Membrane Permeabilization : A Cellular Suicide Stragegy." Doctoral thesis, Linköpings universitet, Experimentell patologi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-11614.
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In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.
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Schneede, Alexander [Verfasser]. "Leben ohne LAMPs : die Folgen des Fehlens der lysosomal assoziierten Membran Proteine LAMP-1 und LAMP-2 auf endosomale, lysosomale Prozesse / Alexander Schneede." Kiel : Universitätsbibliothek Kiel, 2009. http://d-nb.info/1019811161/34.
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Iveson, Graeme Paul. "Passive diffusion across the lysosome membrane." Thesis, Keele University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315231.
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Appelqvist, Hanna. "Lysosomal Membrande Stability and Cathepsins in Cell Death." Doctoral thesis, Linköpings universitet, Experimentell patologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-85008.
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Lysosomes are acidic organelles that are critically involved in a number of physiological processes, including macromolecule degradation, endocytosis, autophagy, exocytosis and cholesterol homeostasis. Several pathological conditions, such as cancer, neurodegenerative disorders and lysosomal storage diseases, involve lysosomal disturbances, indicating the importance of the organelle for correct cellular function. The aim of this thesis was to investigate the role of lysosomes in cell death signaling. Previous studies have shown that permeabilization of the lysosomal membrane and release of hydrolytic enzymes such as cathepsin D to the cytosol occurs during apoptosis. We identified Bid and 14-3-3 proteins as cytosolic targets of cathepsin D in human fibroblasts. Truncated Bid, generated by cathepsin D proteolytic cleavage, stimulates Bax-mediated release of pro-apoptotic factors from the mitochondria, thereby engaging the intrinsic pathway to apoptosis. Since the presence of cathepsins in the cytosol is sufficient to induce apoptosis, the permeability of the lysosomal membrane influences the fate of the cell. In this thesis, we demonstrated that the stability of the lysosomal membrane can be manipulated by altering the lysosomal cholesterol content. Cells with high lysosomal cholesterol content were less prone to undergo apoptosis when challenged with stimuli known to induce lysosome-mediated cell death. In addition, cholesterol accumulation was associated with increased expression of lysosome-associated membrane proteins and storage of other lipids; however, these factors did not contribute to lysosomal stabilization. Lysosomal membrane permeabilization and cathepsins contribute to ultraviolet (UV) irradiation-induced apoptosis. We demonstrate plasma membrane damage induced by UVA irradiation to be rapidly repaired by lysosomal exocytosis in human keratinocytes. Despite efficient plasma membrane resealing, the cells underwent apoptosis, which was dependent on early activation of caspase-8. The activation of caspase-8 was lysosome-dependent and occurred in vesicles positive for lysosomal markers. This thesis demonstrates the importance of lysosomal stability for apoptosis regulation and that this stability can be influenced by drug intervention. Modulation of the lysosomal membrane permeability may have potential for use as a therapeutic strategy in conditions associated with accelerated or repressed apoptosis.
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Lachuer, Hugo. "Role of membrane tension in the spatial regulation of lysosomal exocytosis." Electronic Thesis or Diss., Université Paris sciences et lettres, 2022. http://www.theses.fr/2022UPSLS026.
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L'exocytose lysosomale est impliquée dans plusieurs processus cellulaires, mais sa régulation spatio-temporelle est encore peu connue. En utilisant la microscopie de fluorescence à réflexion totale (TIRFM) et des outils de statistiques spatiales, nous avons observé que l’exocytose aléatoire n’était pas distribuée aléatoirement sur la face ventrale de la membrane plasmique de cellules RPE1, mais organisée en agglomérats sur plusieurs échelles de tailles. Même si le taux d’exocytose est régulé par le cytosquelette d’actine, des perturbations de l’actine ou des microtubules par des drogues n’altèrent pas la structure spatiale de l’exocytose lysosomale. Les événements d’exocytose apparaissent partiellement aux adhésions focales (AF) et leur agglomération est réduite quand la biogenèse des AF est inhibée. De plus, des modifications de la tension de membrane par des chocs hypo-osmotiques et des traitements au méthyl-β-cyclodextrine augmentent l’agglomération des événements d’exocytose. Pour explorer le lien entre AF et tension de membrane, des cellules ont été cultivées sur des micropatrons en forme d’anneau permettant de contrôler l’organisation spatiale des AF. En utilisation une combinaison de TIRFM et de microscopie imageant le temps de vie de fluorescence (FLIM), nous avons pu révéler l’existence d’un gradient de tension radial. En changeant le diamètre du micropatron, nous avons montré que nous pouvions contrôler l’importance de ce gradient et l’agglomération de l’exocytose. En conclusion, nos données indiquent que l’agglomération spatiale de l’exocytose lysosomale repose sur une organisation spatiale de la tension de membrane et la topologie des AFLysosomal exocytosis is involved in many key cellular processes but its spatio-temporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-β-cyclodextrin was found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allows to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM), we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs
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Apfeldorfer, Coralie. "Lysosome biogenesis during osteoclastogenesis." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1164801444532-19433.
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Lysosomes are acidic, hydrolase-rich vesicles capable of degrading most biological macromolecules. During the past several decades, much has been learned about different aspects of lysosome biogenesis. The selective phosphorylation of mannose residues on lysosomal enzymes, in conjunction with specific receptors for the mannose-6-phosphate recognition marker, has been found to be largely responsible for the targeting of newly synthesized lysosomal enzymes to lyzosomes. It is known that lysosomes receive input from both the endocytotic and biosynthetic pathways. Nevertheless the exact molecular mechanisms responsible for sorting of the biosynthetic imput involved in the lysosome biogenesis is still a matter of debate. Because osteoclast precursors do not secrete their lysosomal enzymes and osteoclasts do, the observation of modifications occuring during osteoclastogenesis is a good model to observe mechanisms responsible for lysosomal enzymes traffic. Osteoclasts are bone-degrading cells. To perform this specific task they have to reorganise the sorting of their lysosomal enzymes to be able to target them toward the bone surface in mature cells. Since few years, the differentiation of osteoclasts in vitro did help to study these cells. Osteoclast morphology has been therefore already well studied, and the nature of their specific membrane domains is now established. Sensing the proximity of a bone-like surface the cell reorganises its cytoskeleton, and creates specific membrane domains: an actin-rich ring-like zone (named actin ring) surrounded by highly ruffled membrane (named the ruffled border) where enzymes are secreted, while subsequent bone degradation products are endocytosed. Endocytosed material is then transported through the cell inside transcytotic vesicles and released at the top of the cell in an area named the functional secretory domain. Several molecular machineries are thought to control these different phenomena. The main purpose of this thesis was to identify the major regulators of lysosomal enzymes secretion and therefore to identify the molecular switches responsible for such a membrane traffic re-organisation.
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Kaur, Jasber. "Targeting of membrane proteins to lysosome-related organelles." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397316.
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Kuwana, Tomomi. "Characterisation of a lysosomal protein that interfrers with membrane fusion assays." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337875.
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Cordier, Guillaume. "Unravelling motor protein organization on lysosomal membranes with super-resolution microscopy." Doctoral thesis, Universitat Politècnica de Catalunya, 2018. http://hdl.handle.net/10803/565415.
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This thesis first develops new methods for high-throughput and multi-color super-resolution microscopy (Chapters 2 and 3). Subsequently, I apply these methods to study the organization of motor proteins on the lysosome membrane inside cells with the purpose of determining how intracellular transport can be regulated via motor-protein organization (Chapter 4). Chapter 1 is an Introduction to the state of the art for our knowledge in microtubule-based intracellular transport. Chapter 2 introduces the single molecule localization techniques that improve the spatial resolution of light microscopy. This chapter emphasizes the Stochastic Optical Reconstruction Microscopy (STORM) technique, which I used to study the organization of microtubule based motor proteins around lysosomes as well as the fusion and fission of mitochondria. Chapter 3 describes the development of two new techniques: (i) the use of microfluidic devices to improve the throughput of correlative live-cell and super-resolution microscopy, thus allowing to observe rare events and (ii) sequential multi-color imaging that increases the number of colors that can be imaged with STORM. Chapter 4 focuses on the biological application of sequential multicolor imaging to study the 3D organization of dynein and kinesin on lysosomal membranes. Conclusions and Future Perspectives are provided in Chapter 5.El transporte intracelular es de vital importancia para mantener la organización subcelular. Fallos en el transporte intracelular pueden desembocar en diversas enfermedades, especialmente si el fallo tiene lugar en el sistema nervioso. Los actores principales involucrados en el transporte intracelular son el citoesqueleto de microtúbulos y las proteínas motoras. El citoesqueleto de microtúbulos conecta la región perinuclear a la periferia de la célula, actuando así como carriles para el transporte. Las proteínas motoras se desplazan a lo largo del citoesqueleto de microtúbulos para llevar cargas. Hay dos tipos de familias de proteínas motoras que desplazan cargas a lo largo de los microtúbulos polarizados: la superfamilia de las kinesinas se mueve hacia el extremo positivo del carril microtubular mientras que las dineínas se mueven hacia el extremo negativo del carril microtubular. Desde su descubrimiento en los años sesenta, las proteínas motoras han sido extensamente estudiadas usando métodos in vitro de una sola molécula, permitiéndonos entender las bases del transporte intracelular. No obstante, las proteínas motoras, sus carriles microtubulares y las vesículas que portan tienen tamaños comprendidos de decenas a centenas de nanómetros, por debajo del límite de difracción de la microscopía óptica. Así, el estudio del tráfico intracelular en el contexto celular ha sido desafiante. Mientras que los estudios in vitro son altamente valiosos para mejorar nuestra comprensión de cómo funcionan las proteínas motoras, no pueden capturar la complejidad total del ambiente intracelular. Por tanto, es muy importante desarrollar nuevos métodos para superar el desafío de estudiar el transporte intracelular en células intactas. En la última década, el desarrollo y la llegada de los métodos de microscopía de superresolución, ha supuesto una revolución en el campo de la microscopía óptica. Estos métodos han hecho posible visualizar estructuras subcelulares con resolución espacial nanométrica, rompiendo el límite de difracción. Esta tesis primero desarrolla nuevos métodos para microscopía de alto rendimiento y superresolución multicolor (Capítulos 2 y 3). Seguidamente, aplico estos métodos para estudiar dentro de las células la organización de las proteínas motoras en la membrana del lisosoma con el objetivo de determinar cómo el transporte intracelular puede estar regulado a través de la organización de las proteínas motoras (Capítulo 4). El Capítulo 1 es una introducción al estado de la técnica para nuestro conocimiento en el transporte intracelular basado en microtúbulos. El Capítulo 2 introduce las técnicas de localización de una sola molécula que mejoran la resolución espacial de la microscopía óptica. Este capítulo enfatiza sobre la técnica de Microscopía de Reconstrucción Óptica Estocástica (STORM), que utilicé para estudiar la organización de las proteínas motoras interactuando con microtúbulos, encima de los lisosomas, así como la fusión y la fisión de las mitocondrias. El Capítulo 3 describe el desarrollo de dos nuevas técnicas: (i) el uso de dispositivos microfluídicos para mejorar el rendimiento de la tecnica de correlacion entre celulas vivas y la microscopía de superresolución permitiendo observar evento raros y (ii) imágenes secuenciales multicolor que incrementan el número de colores que pueden ser fotografiados con STORM. El Capítulo 4 se centra en la aplicación biológica de las imágenes secuenciales multicolor para estudiar la organización tridimensional de la dineína y kinesina en la membrana lisosomática. Las conclusiones y perspectivas de futuro se presentan en el Capítulo 5.
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Ebrahim, Roshan. "Biogenesis of lysosomes in macrophages : intracellular pathway of lysosomal membrane protein to lysosomes." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/3126.
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Foster, Willam. "The effects of lysosomal Ca2+ release on membrane depolarisation and synaptic plasticity in hippocampus." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:044cb37b-fdb7-4c19-b6f7-c9e261817751.
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Intracellular Ca2+ signalling is essential for the control of almost every physiological process, from muscular contraction to synaptic transmission. Intracellular Ca2+ signals can be generated from both extracellular or intracellular Ca2+ stores. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous and important Ca2+ mobilising second messenger. NAADP signalling causes Ca2+ release from intracellular acidic Ca2+ stores. The functional roles of NAADP signalling and acidic store Ca2+ release in the central nervous system are relatively unknown Brailoiu et al. (2009b) showed that NAADP signalling enhances membrane excitability in neurons of the medulla, Padamsey and Emptage (2011) (Unpublished) find similar effects in pyramidal neurons of the hippocampus. I used pharmacological manipulations in combination with electrophysiological techniques and dendritic Ca2+ imaging to explore the effect of NAADP/acidic store signalling on membrane excitability and synaptic plasticity in pyramidal neurons of the hippocampus. I began by using a membrane permeable form of NAADP (NAADP-AM) to show NAADP/acidic store Ca2+ signalling caused membrane depolarisation. I also showed, with intracellular dialysis of Ca2+ mobilising second messengers, NAADP is unique in its ability to directly cause membrane depolarisation. I then identified glutamate, acting via metabotropic glutamate receptor 1 (mGluR1), as an endogenous stimulus that causes NAADP-mediated Ca2+ release and depolarisation. I next elucidated the signalling pathway responsible for mGluR1/NAADP-mediated depolarisation and showed the requirement of acidic store Ca2+ release, and subsequent amplification of this Ca2+ via ryanodine receptors by Ca2+-induced Ca2+ release. The resulting Ca2+ signal caused inhibition of small conductance K+ channels (SK channels) and membrane depolarisation. SK channels are described to facilitate the induction of plasticity in hippocampal synapses by modulation of GluN Ca2+ entry (Ngo-Anh et al., 2005). Finally, I show that the induction of mGluR1-dependent long-term potentiation requires inhibition of the SK channels via NAADP/acidic store Ca2+ signalling. Group 1 mGluRs are implicated in the pathogenesis of neurodevelopmental disorders such as fragile X syndrome. My findings may identify new targets for the treatment of such diseases.
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Crombie, Andrea Rene. "Lysosomal integral membrane protein II, a member of the CD36 gene family : comparative analysis of structure-function relationships /." Access full-text from WCMC, 1998. http://proquest.umi.com/pqdweb?did=733079741&sid=9&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Steenhuis, Pieter [Verfasser], and Stephan [Akademischer Betreuer] Storch. "Intracellular Sorting and Biochemical Analysis of the Lysosomal Membrane Protein CLN7 / Pieter Steenhuis. Betreuer: Stephan Storch." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/1069376825/34.
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Danaher, Michelle [Verfasser]. "Characterisation of the Lysosomal Integral Membrane Protein Type 2 (LIMP‐2)in Murine Brain / Michelle Danaher." Kiel : Universitätsbibliothek Kiel, 2017. http://d-nb.info/1137509708/34.
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Ecard, Jason. "Mécanismes de tri et voies de transport intracellulaire des protéines de la membrane du lysosome." Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS094.pdf.
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Plus d’une soixantaine de protéines différentes sont insérées dans la membrane du lysosome et participent aux différentes fonctions de cet organite. Mais les voies de transport intracellulaire qui mènent les protéines membranaires lysosomales (LMP) jusqu’à cet organite ne sont pas bien comprises. De façon intéressante, certaines LMP présentent des localisations intracellulaires anormales dans certains cancers. C’est notamment le cas de la glycoprotéine LAMP1 qui s’avère surexposée à la surface cellulaire dans plusieurs cancers, où elle y joue plusieurs rôles dans l’agressivité tumorale. Grâce au système RUSH (pour Retention Using Selective Hooks), permettant la synchronisation du transport le long de la voie de sécrétion, nous avons montré que LAMP1, après néosynthèse, passe par la membrane plasmique avant d’accéder aux endosomes. La comparaison des voies empruntées par différentes LMP a aussi permis de révéler que LAMP1 et LIMP2 sont triées au niveau de l’appareil de Golgi, LIMP2 étant concentrée dans des structures vésiculaires caractéristiques et dépourvues de clathrine. Nous avons aussi montré que, de façon surprenante, ce tri au niveau de l’appareil de Golgi est indépendant des signaux de recrutement d’adaptateurs à la clathrine portés dans les queues C-terminales de ces deux LMP. Afin d’étudier quels mécanismes pourraient être impliqués dans la surexposition de LAMP1 à la surface cellulaire de certains cancers, nous avons aussi réalisé un criblage d’inactivation de gènes basé sur la technologie CRISPR-Cas9. Nous avons sélectionné les gènes dont l’inactivation affectait les niveaux de LAMP1 en surface et nous avons obtenu de nombreux gènes candidats qui sont à l’étudeMore than sixty different proteins are inserted into the membrane of the lysosome, participating in the various functions of this organelle. But the intracellular trafficking pathways that lead lysosomal membrane proteins (LMPs) to this organelle are not well understood. Interestingly, some LMPs exhibit abnormal intracellular localisations in some cancers. This is the case of the glycoprotein LAMP1 which is overexpressed at the cell surface in several cancers, from where it plays several roles in tumour aggressiveness. Thanks to the Retention Using Selective Hooks (RUSH) system, allowing the synchronisation of the transport along the secretory pathway, we have shown that neosynthesized LAMP1 reaches the plasma membrane before entering endosomes. Comparing the routes followed by different LMPs also revealed that LAMP1 and LIMP2 are sorted at the Golgi apparatus, LIMP2 being concentrated in characteristic vesicular and clathrin-free structures. We have also shown that, surprisingly, this sorting at the level of the Golgi apparatus is independent of clathrin adapters recruitment signals carried in the C-terminal tails of these two LMPs. To investigate what mechanisms might be involved in the overexposure of LAMP1 at the cell surface of certain cancers, we also performed a gene knock-out screen based on CRISPR-Cas9 technology. We selected genes whose inactivation affected LAMP1 levels at the surface and obtained many candidate genes that are under study
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Stringer, Daniel Kenneth. "The role of ubiquitination within the endocytic pathway." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/2775.
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Ubiquitination is a post-translational modification tht mediates sorting of integral membrane proteins to lysosomes for their degradation. ESCRTs (Endosomal Sorting Complex Required For Transport) bind and sequester ubiquitinated membrane proteins and direct them into multivesicular bodies (MVBs). ESCRTs themselves become covalently ubiquitinated, simply by virtue of non-covalently binding Ub. However, it is unclear whether this regulates a critical aspect of ESCRT function. In yeast, many MVB cargo proteins are ubiquitinated by the HECT-type Ub-ligase Rsp5, sometimes via the action of Rsp5 adaptor proteins. While many Rsp5 targets are modified by polyubiquitination, it remains unclear whether polyubiquitination is a necessary signal for their incorporation into MVBs. Despite years of research, these and related questions have been difficult to resolve because it is technically quite challenging to control the level of a given protein's ubiquitination. The aim of this research was to develop a novel technique, which can render proteins resistant to ubiquitination. The technique involved the fusion of the Ub-peptidase to a protein of interest via a flexible linker, essentially creating a "DUb module". The intent of this module would be to cleave any Ub form the target protein, essentially immunizing it from the effects of ubiquitination. This novel method was used in combination with several conventional methods to examine the role of ubiquitination within the endocytic pathway and in particular focus on the questions of what type of ubiquitin signal was sufficient for sorting into MVB vesicles and whether ubiquitination of ESCRTs was required for their sorting activity. We found that a single Ub was sufficient for membrane protein entry into MVBs in the absence of ESCRT ubiquitination.
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Bell, Catherine M. "RHEB DYNAMICS ON LYSOSOMAL MEMBRANES DETERMINES MTORC1 ACTIVITY AFTER LOSS OF P53 OR ACTIVATION OF AMPK." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/4036.
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The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss or mutation of p53. This activation involves the loss of TSC2 from lysosomal membranes, the site of mTORC1 activation by Rheb. We demonstrate that loss of lysosomal TSC2 increased the levels of lysosomal Rheb. Control of mTORC1 was restored by overexpression of TSC2, which correlated with decreased lysosomal Rheb. Surprisingly, pemetrexed-activated AMPK did not phosphorylate TSC2 because of an accumulation of nonfunctional p53, and a subsequent decrease in TSC2 mRNA. Accordingly, lysosomal TSC2 decreased, however, the levels of lysosomal Rheb decreased. Future studies will question whether the robust Raptor phosphorylation by pemetrexed is involved in this decrease in lysosomal Rheb. AMPK activation by pemetrexed also significantly increased the translocation of AMPK to the nucleus, and we will explore the function of this nuclear AMPK. Overall, these findings present a mechanism involved in the oncogenic signaling of mTORC1 with loss of p53 and offer insight into how pemetrexed reinstates control.
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Alvarez, Valadez Karla. "Targeting intracellular cholesterol transport for inducing lysosomal damage and immunogenic cell death in cancer." Electronic Thesis or Diss., université Paris-Saclay, 2023. http://www.theses.fr/2023UPASL123.
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Les lysosomes jouent un rôle central dans la régulation des processus anaboliques et cataboliques, la signalisation cellulaire ainsi que dans la mise en œuvre des programmes transcriptionnels au sein des cellules. Ils favorisent l’adaptation des cellules cancéreuses lors des variations du microenvironnement en leur fournissant les métabolites essentiels et l’énergie nécessaire à leur survie et à leur prolifération. Un acteur majeur dans la réponse adaptative des lysosomes est le facteur de transcription EB (TFEB). TFEB coordonne l’expression de gènes associés à la fonction et à la biogenèse des lysosomes, y compris ceux impliqués dans l’autophagie, un processus catabolique majeur des cellules qui dépend des lysosomes. TFEB et l’autophagie fonctionnent comme des mécanismes adaptatifs visant à rétablir l’homéostasie cellulaire en réponse à un stress. Cependant, la biogenèse des lysosomes et l'augmentation de leur taille induite par TFEB peuvent rendre les cellules cancéreuses plus vulnérables aux composés ciblant les lysosomes. Cette vulnérabilité ouvre la porte au développement de nouvelles stratégies pour lutter contre le cancer en ciblant simultanément les lysosomes et en activant TFEB. L’objectif initial de cette étude a été de découvrir de nouveaux agents pharmacologiques agonistes de TFEB, manifestant une cytotoxicité significative contre les cellules cancéreuses. Par un criblage de la bibliothèque Prestwick comprenant 1200 composés approuvés par la « Food and Drug Administration » (FDA), nous avons identifié deux antidépresseurs, la sertraline et l’indatraline, qui agissent en tant que puissants activateurs de la translocation de TFEB vers le noyau. Les deux composés induisent une accumulation de cholestérol au sein des lysosomes, entraînant la perméabilisation de leurs membranes et une perturbation du flux autophagique. L’analyse de modélisation moléculaire a révélé que les deux composés pourraient inhiber le trafic du cholestérol en se liant au site de fixation du cholestérol des transporteurs, Niemann-Pick type C1 (NPC1) et NPC2. Dans les cellules cancéreuses, la sertraline et l’indatraline provoquent une mort cellulaire immunogénique, en transformant les cellules mourantes en vaccins prophylactiques capables de protéger contre la croissance tumorale chez la souris. Dans un contexte thérapeutique, une dose unique de ces composés était suffisante pour ralentir de façon significative la croissance tumorale de manière dépendante des lymphocytes T. Ces résultats caractérisent la sertraline et l’indatraline comme des agents immunostimulants qui agissent à travers un mécanisme novateur connectant l’accumulation du cholestérol lysosomal aux dommages lysosomaux, entraînant ainsi la mort immunogénique des cellules cancéreuses. Ces résultats soutiennent le repositionnement de ces deux molécules en tant qu’agents immunostimulants pour le traitement du cancer et encouragent l’extension de cette étude à d’autres inhibiteurs du transport lysosomal du cholestérolLysosomes serve as an intracellular platform that coordinates anabolic and catabolic processes, cell signaling, and transcriptional programs. These organelles allow the adaptation of cancer cells to a changing microenvironment by supplying them with essential metabolites and energy for their survival and proliferation. A major player in the lysosomal adaptive response is the transcription factor EB (TFEB), which is part of the microphthalmia/transcription factor E (MIT/TFE) family of transcription factors. TFEB plays a pivotal role in driving the expression of several genes associated with lysosome function and biogenesis, including those participating in autophagy. The latter is a critical lysosomal catabolic process in the cell. While TFEB and autophagy function as adaptive mechanisms to reestablish cellular homeostasis in response to stressors, TFEB-induced lysosomal biogenesis and enlargement can render cancer cells more vulnerable to compounds targeting lysosomes. This vulnerability opens the door for developing new strategies to combat cancers by simultaneously targeting the lysosome and activating TFEB. This study initially aimed to uncover novel pharmacological agents that function as agonists of TFEB and exhibit substantial cytotoxicity against cancer cells. By conducting cell-based drug screening of the Prestwick library, consisting of 1200 Food and Drug Administration (FDA)-approved compounds, we identified two antidepressants, sertraline and indatraline, as potent inducers of TFEB nuclear translocation. Both compounds promoted cholesterol accumulation within lysosomes, resulting in lysosomal membrane permeabilization, disruption of autophagy, and cell death. Molecular docking analysis unveiled that indatraline and sertraline may inhibit cholesterol traffic by binding to the same cavity where cholesterol typically binds to the lysosomal cholesterol transporters, Niemann-Pick type C1 (NPC1) and NPC2. In cancer cells, sertraline and indatraline elicited immunogenic cell death, converting dying cells into prophylactic vaccines that were able to protect against tumor growth in mice. In a therapeutic setting, a single dose of each compound was sufficient to significantly reduce the outgrowth of established tumors in a T cell-dependent manner. These results identify sertraline and indatraline as immunostimulatory agents that operate through a novel mechanism that connects lysosomal cholesterol accumulation to lysosomal membrane permeabilization, ultimately leading to immunogenic cell death. These results support the repositioning of sertraline and indatraline as immunostimulatory agents for cancer treatment and encourage the broadening of this study to other lysosomal cholesterol transport inhibitors
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Kongmanas, Kessiri. "Roles of Seminolipid and Its Associated Membrane Domain in Male Fertility." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32509.
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Our research aims at understanding the roles of seminolipid (sulfogalactosylglycerolipid or SGG) and its associated membrane domains in male reproduction. SGG is a sulfoglycolipid present selectively and abundantly in mammalian male germ cells. Therefore, information on its properties would be relevant towards the development of male fertility biomarkers and spermicide-based contraceptives. We have shown that SGG has direct affinity for zona pellucida (ZP, egg extracellular matrix) and plays a role in the formation of sperm lipid rafts, the ZP-binding platforms on the sperm anterior head plasma membrane (APM), the initial ZP binding site. For a better understanding of mechanisms underlying sperm-ZP interaction, I performed proteomic characterization of APM vesicles (SGG-associated membrane domains with ZP affinity) isolated from sperm before and after capacitation, a process through which sperm gain maximal ZP affinity. Proteomic results revealed that capacitated APM vesicles contained high-molecular-weight protein complexes, with higher ZP affinity and levels of ZP-binding proteins as compared with those of the non-capacitated samples. ZP-binding proteins known to exist in the acrosome (i.e., zonadhesin, proacrosin/acrosin) were found in these APM protein complexes. Immunofluorescence suggested that a fraction of these proteins trafficked from the acrosome to APM during capacitation. These findings provided a new mechanism on how sperm gain full ZP-binding ability during capacitation. Since SGG is a major component of APM, proper SGG levels at this site would be important for male fertility. Levels of sperm SGG are regulated through the synthesis and degradation. In fact, lack of SGG-synthesis enzymes causes a spermatogenesis disruption, resulting in male infertility. However, significance of SGG degradation remains unknown. SGG can be desulfated in vitro by arylsulfatase A (ARSA), an enzyme existing in the acrosomes of sperm/spermatids and lysosomes of Sertoli cells, testicular somatic cells that nurture developing germ cells. Sertoli cells also phagocytose ~50% of germ cells that become apoptotic during spermatogenesis. To understand physiological importance of SGG degradation, the fertility status and SGG levels of Arsa-/- male mice were determined. We found that Arsa-/- males became subfertile when they were older than 5 months, and when they were 8-month-old (~40-year-old men) they produced sperm at 50% wild type rate. Arsa-/- sperm had minimal in vitro fertilizing ability and a number of them showed abnormal morphology. Quantitative mass spectrometry revealed that SGG levels in Sertoli cells of 8-month-old Arsa-/- mice were increased to ~250% of the wild type level; this SGG accumulation may lead to a decrease in Sertoli cell ability to support spermatogenesis. However, SGG levels in sperm of 8-month-old Arsa-/- mice were ~50% of the wild type value, a result that partly explained the decreased fertilizing ability of these sperm. The reduced SGG level of Arsa-/- sperm was likely due to a lack of SGG’s building-block lipid (palmitylpalmitoylglycerol) putatively generated in Arsa-/- Sertoli cells and recycled to the next generation of primary spermatocytes for SGG synthesis. Hence, levels of sperm SGG are a promising bioindex for male fertility. Since Sertoli cells also regulate SGG homeostasis, their functionality should be now included in male fertility/subfertility diagnosis.
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Chandramohanadas, Rajesh. "Rapid purification of human lysosomal membranes, characterisation of the detergent resistant microdomains, purification and reconstitution of the vacuolar proton pump (V-ATPase)." [S.l.] : [s.n.], 2006. http://archiv.ub.uni-marburg.de/diss/z2006/0240.
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Pasquier, Adrien. "Lysosomal degradation of insulin granules promotes β-cell failure in type 2 diabetes." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ083/document.
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Notre équipe a récemment découvert l’importance du ciblage des granules d’insuline aux lysosomes lors d’une mise à jeun chez les cellules pancréatiques β. Le diabète de type 2 (TD2) est caractérisé par la résistance à l’insuline couplé au dysfonctionnement des cellules β-et à leur perte. Je souhaitais évaluer le ciblage des granules d’insuline aux lysosomes dans le contexte diabétique. Grâce à un modèle murin, nous avons trouvé que le nombre des lysosomes contenant des granules d’insuline était augmenté chez les cellules β-provenant de souris diabétiques en comparaison aux contrôles. Ceci était accompagné par l’augmentation des niveaux de la protéine lysosomale CD63. Parce que PKD1 contrôle le ciblage des granules d’insuline aux lysosomes lors d’une mise à jeun, nous nous sommes demandé si PKD1 était importante lors d’un diabète de type 2. Dans nos modèles, les niveaux de PKD1 étaient diminués en conditions diabétiques en comparaison aux contrôles. De plus, l’inhibition de PKD1 entrainait l’augmentation du ciblage des granules d’insuline aux lysosomes et accélérait l’apparition du diabète dans notre modèle murin. Nous souhaitions ensuite savoir si l’activation de PKD1 dans les cellules pancréatiques β-pouvait être avantageuse dans un contexte diabétique. De fait, grâce à l’utilisation d’un composé spécifique, nous avons pu montrer que l’activation de PKD1 menait à l’augmentation des niveaux d’insuline sur des ilots pancréatiques humains et ralentissait l’apparition du diabète dans notre modèle murin. Pour conclure, j’ai aussi débuté la caractérisation des lysosomes sur d’autres types cellulaires des ilots pancréatiques. Nous avons observé que LIMP2, une autre protéine lysosomale, était fortement exprimée chez les cellules pancréatiques αOur team recently uncovered the importance of the targeting of insulin granules to the lysosomal compartments in pancreatic β-cells during fasting. Type 2 Diabetes (T2D) is characterised by insulin resistance coupled with pancreatic β-cell failure which account for both β-cells dysfunction and β-cells death. I wanted to assess the targeting of insulin granule to the lysosomes in the context of T2D. Using murine diabetic model, we found that the number Granule-containing Lysosomes was enhanced in diabetic β-cells in comparison to controls. This was accompanied by an increase in the level of the lysosomal protein CD63. Because PKD1 controls the targeting of insulin granule to the lysosomes during fasting, I wondered if PKD1 was important during T2D. PKD1 levels were decreased in our diabetic models in comparison to controls. Moreover inhibition of PKD1 led to enhanced targeting of the insulin granules to the lysosomes and accelerated apparition of diabetes in our murine model. I also tested if activation of PKD1 in pancreatic β-cells could be beneficial in the context of diabetes. Indeed using a specific compound, we showed that PKD1 activation led to an increase in insulin levels and delayed onset of diabetes in our murine model. My work thus uncovered mechanisms underlying a fundamentally new process in β-cells with potential implications for novel therapeutic directions in T2D. Finally, I started to assess lysosomes in another pancreatic islets cell type. I found that LIMP2, another lysosomal membrane protein, was specifically highly expressed in the pancreatic α-cells
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Zunke, Friederike [Verfasser]. "Characterisation of the Lysosomal Integral Membrane Protein Type-2 (LIMP-2) and its interaction with b-Glucocerebrosidase: implications for Parkinson and Gaucher Disease / Friederike Zunke." Kiel : Universitätsbibliothek Kiel, 2018. http://d-nb.info/1172288046/34.
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Cunningham, Dawn Anne. "Human lysosomal associated membrane protein-2 (LAMP-2) : a molecular bridge between infection and autoimmunity in an animal model of focal necrotising and crescentic glomerulonephritis (FNCGN)." Thesis, University of Aberdeen, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485684.
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Pauci-immune focal necrotising and crescentic glomerulonephritis (FNCGN) is a severe form of kidney disease often associated with anti-neutrophil cytoplasmic antibodies (ANCA). Well pocumented . ANCA target proteins . include myeloperoxidase (MPO) and proteinase 3 (PR3), both contained in the Iysosomes of neutrophils. A study by Kain et al (1995) identified a novel ANCA target; lysosomal associated membrane protein 2 (LAMP-2) (Kain et aI., 1995) recognised by FNCGN patient sera. An immunodominant epitope shares sequence homology with a peptide found on bacterial adhesion protein fimH (Kain et aI., Manuscript in Preparation). The aim of this work has been to evaluate the pathogenic potential of cross reactive antibodies that bind to both LAMP-2 and fimH to provide an explanation of how persistent infection can drive chonic inflammatory responses that underpin autoimmune disease. A WKY rat model of FNCGN was used to examine this concept of molecular mimicry by using both passive and active immunisation schedules. Recombinant fragments of human and rat LAMP-2 and bacterial fimH were generated and purified ·following expression in plasmid vectors. The WKY rat model was first passively immunised with rabbit antibodies specific for human LAMP-2 (hLAMP-2) to confirm that they had the ability to cross-react with sequence homologous rat LAMP-2 (rLAMP-2). Indirect immunofluorescence (IIF) and ELISA confirmed cross reactivity and further indicated that antihLAMP- 2 resulted in tissue damage similar to that observed in human FNCGN. Further, active immunisation with hLAMP-2 in the presence of adjuvant was sufficient to break tolerance, generating a pathogenic antibody response specific for both human and rat LAMP-2.
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Bodammer, Beatrice [Verfasser], Ralf [Akademischer Betreuer] Ficner, Kurt von [Akademischer Betreuer] Figura, and Hans-Joachim [Akademischer Betreuer] Fritz. "Untersuchung der Acetyl-Coenzym A:alpha-Glucosaminid N-Acetyltransferase in gereinigten lysosomalen Membranen / Beatrice Bodammer. Gutachter: Kurt von Figura ; Hans-Joachim Fritz. Betreuer: Ralf Ficner." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2012. http://d-nb.info/1043029028/34.
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Qu, Yan. "Regulation of Multiple Membrane Trafficking Pathways Stimulated by P2X7 Receptor Activation in Inflammatory Macrophages." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1221066011.
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Ziegler, Paul K. [Verfasser], Florian R. [Akademischer Betreuer] Greten, Thomas [Gutachter] Kirchner, Thomas [Gutachter] Korn, and Bernhard [Gutachter] Holzmannn. "Stat3 Prevents Mitophagy and Lysosomal Membrane Permeabilization in Intestinal Epithelial Cells to Suppress Adaptive Immunity during Tumorigenesis / Paul K. Ziegler ; Gutachter: Thomas Kirchner, Thomas Korn, Bernhard Holzmannn ; Betreuer: Florian R. Greten." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1181326338/34.
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Strathmann, Marc. "Auswirkungen der Bestrahlung mit UVB, UVA-1 und PUVA-1 auf das Funktionsverhalten humaner dermaler Mastzellen nach Stimulation mit anti-IgE und Substanz P." Doctoral thesis, [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972615822.
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Atakpa, Peace. "Ca2+ signalling between the endoplasmic reticulum and lysosomes." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288002.
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Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.
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Wenham, Matt. "The role of Adaptor Protein 3 in cytotoxic T lymphocytes." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:1a5c1fd6-c8cc-454f-81a5-b9a5a18c1540.
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Cytotoxic T lymphocytes (CTL) kill virally infected and tumourigenic cells via the regulated secretion of specialised secretory lysosomes. These secretory lysosomes contain cytolytic effector molecules, such as perforin and granzymes, which are able to induce apoptosis in target cells. Secretion occurs at the contact point between the CTL and its target, in a highly structured region termed the immunological synapse (IS). Upon formation of the IS, CTL undergo polarisation of their microtubule cytoskeleton and movement of the microtubule organising centre (MTOC) to the IS. Secretory lysosomes are then able to polarise along microtubules, fuse with the plasma membrane and deliver their effector molecules to the IS. The Adaptor Protein 3 complex (AP-3) sorts transmembrane proteins to lysosomes and deficiency in AP-3 results in missorting of proteins from the lysosomal to plasma membrane. CTL from AP-3 deficient patients, who suffer from Hermansky-Pudlak Syndrome Type 2 (HPS2), show reduced killing of target cells. This thesis describes two new patients with HPS2, both with homozygous mutations in the AP3B1 gene, which codes for the β3A subunit of the AP-3 complex. CTL from the new HPS2 patients show reduced cytotoxicity, which is shown here to be due to impaired secretory lysosome polarisation towards the IS. This impairment is common to HPS2 CTL, but varies between patients. In order to determine differences between HPS2 and wild type CTL, the localisation of a range of lysosomal, cytolytic, transmembrane, inhibitory and activation marker proteins is examined. This shows that in HPS2 CTL, LAMP1, CD63 and CD9 are potential AP-3 cargos. In addition, a possible effect on the key lytic effector perforin is identified. Preliminary experiments to allow proteomic comparison of HPS2 and wild type CTL are also presented. Further investigation of these results will help to shed light on the mechanisms involved in secretory lysosome polarisation in CTL.
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Tejle, Katarina. "Leishmania donovani Lipophosphoglycan : Modulation of Macrophage and Dendritic Cell Function." Doctoral thesis, Linköping : Univ, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-6527.
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Rizzato, Vanessa Rodrigues. "Envolvimento da neuraminidase-1 na atrofia muscular." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5138/tde-01122014-094857/.
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Sialidose é uma doença neurossomática causada pela deficiência congênita da neuraminidase-1 (Neu1), enzima envolvida na regulação do catabolismo de sialoglicoconjugados nos lisossomos. Com o acúmulo de sialoglicoconjugados, ocorre comprometimento sistêmico e neurológico. Achados histológicos musculares incluem expansão da matriz extracelular (MEC) devido à proliferação anormal de fibroblastos, invasão das fibras musculares por componentes da MEC, fragmentação do citoplasma, formação vacuolar e atrofia das fibras musculares. Entretanto o mecanismo da atrofia muscular na deficiência de Neu1 não está completamente esclarecido, sendo o objetivo desse estudo. Desnervou-se o músculo gastrocnêmio direito de camundongos com deficiência de Neu1 (Neu1 -/-) e de controles Neu1 +/+. Os animais foram eutanasiados 0, 3, 7, 14 e 21 dias pós desnervação. Os músculos desnervados e contralaterais foram submetidos às seguintes análises: 1) histologia geral e medida da área transversa das fibras; 2) autofagia, através da avaliação da presença de vacúolos autofágicos por estudo ultraestrutural e da análise da expressão da proteína LC3; 3) ativação do sistema lisossomal, por reação de fosfatase ácida e análise da expressão proteica de catepsina L e lamp1; 4) deposição de colágeno e infiltração de tecido conjuntivo no tecido muscular; 5) níveis das proteínas Akt e GSK3b; 6) expressão dos atrogenes MuRF1 e Atrogina-1; 7) níveis da proteína MyoD, relacionada à diferenciação muscular; e 8) expressão dos genes Neu1, Neu2, Neu3 e Neu4. Os animais Neu1-/- apresentaram menor peso corporal e muscular compararando-se com animais Neu1 +/+. Houve redução progressiva da área das fibras dos músculos desnervados em relação aos músculos contralaterais. Os animais Neu1-/- apresentaram atrofia muscular basal, com aumento acentuado dos espaços endomisiais e perimisiais. Ocorreu formação de vacúolos autofágicos a partir de 14 dias de desnervação tanto em animais Neu1+/+ quanto em Neu1-/-. Os níveis de expressão proteica de catepsina L e de lamp1 aumentaram a partir de 14 dias de desnervação, mais notadamente em músculos desnervados de camundongos Neu1-/-. A expressão proteica de colágeno III mostrou-se aumentada em animais Neu1-/-, principalmente após desnervação. A expressão proteica da forma fosforilada do Akt (forma ativada) diminuiu após 21 dias de desnervação principalmente em músculos desnervados de animais Neu1+/+. Os níveis de PGSK3 b, forma inativa de GSK3b, diminuíram após a desnervação, em animais Neu1+/+ e animais Neu1-/-. Houve aumento na expressão gênica de Atrogina-1 e MuRF1 após 3 e 7 dias de desnervação, respectivamente; a expressão gênica de Atrogina-1 nos camundongos Neu1-/- teve um aumento atrasado, mostrando diferença significante após 7 dias de desnervação. Não houve diferença significativa entre níveis proteicos de MyoD. A expressão gênica de Neu1 mostrou-se elevada em músculos desnervados de animais Neu1+/+. Conclui-se, portanto, que a Neu1 parece atuar na regulação da massa muscular principalmente controlando o processo de ativação do sistema lisossomal, porém aparentemente sem afetar a autofagiaSialidosis, a severe neurosomatic disease, results from congenital neuraminidase-1 (Neu1) deficiency. This enzyme regulates the catabolism of sialoglycoconjugates in the lysosomes. Systemic and neurologic manifestations occur due to the sialoglycoconjugates accumulation. In the mouse model for Neu1 deficiency, the muscle histologic findings include extracellular matrix (ECM) expansion, due to abnormal fibroblast proliferation, muscle fibers invasion by ECM components, cytoplasm fragmentation, vacuolar formation and muscle atrophy. Nevertheless the mechanisms of muscle atrophy in Neu1 deficiency are not completely known. This study was designed to investigate Neu1 involvement in muscle atrophy process. Denervation of gastrocnemius muscle was performed by sectioning sciatic nerve from Neu1 deficient mice (Neu1 -/-) and from normal control Neu1 +/+; the animals were euthanized 0, 3, 7, 14 and 21 days after denervation. Denervated and control muscles were collected and submitted to several analysis: 1) histological; 2) autophagic vacuoles formation, performed by ultrastructural analysis and LC3 protein expression; 3) acid phosphatase reaction, lamp1 and cathepsin L protein expression, to analyze lysosomal activation; 4) collagen deposition and fibrous formation; 5) proteins involved with muscle trophism, Akt and GSK3b; 6) MuRF1 and Atrogin-1 gene expression; 7) MyoD protein expression; 8) Neu1, Neu2, Neu3 and Neu4 genes expression. Neu1 -/- mice presented decreased body and muscle weight comparing to Neu1 +/+ animals. Muscle fiber cross-sectional area was reduced in denervated muscles comparing to contralateral muscles. Neu1 -/- mice muscles presented basal atrophy and increase of endomisial and perimisial spaces, which became more evident after denervation. After 14 days of denervation, autophagosome formation was noticed on Neu1 +/+ and Neu1-/- animals. Cathepsin L protein levels were increased after 14 and 21 days of denervation, especially in denervated muscles from Neu1 -/- mice. Lamp1 protein expression was increased in Neu1-/- animals. Type III collagen protein levels were increased in Neu1-/- animals. There were no significant differences between MyoD protein levels. P-Akt, active form of Akt protein levels, decreased after 21 days of denervation, especially in denervated muscles from control group animals, indicating that protein synthesis is decreased. P-GSK3b, inactive form of GSK3b decreased in denervated muscles from Neu1 -/- and Neu1 +/+ animals, which indicates that this protein remained activated during muscle atrophy process. There were significant differences in Atrogin-1 and MuRF1 gene expression levels after 3 and 7 days of denervation. Neu1 -/- animals muscles presented a delayed Atrogin-1 response. Neu1 gene expression was increased in denervated muscles from Neu1 +/+ mice. These findings suggest that Neu1 seems to act in the regulation of muscle mass mainly by controlling the process of lysosomal system activation, but apparently without affecting autophagy
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Lang, Thierry. "Etude des interactions macrophage-bacterie : cinetique des communications entre le phagosome contenant des bacteries pathogenes ou non pathogenes et les autres compartiments membranaires impliques dans l'endocytose." Paris 7, 1987. http://www.theses.fr/1987PA077126.
Full textAbstract:
Determiner si la pathogenicite de microbacterium avium s'exprime par une alteration du processus d'endocytose chez le macrophage de la moelle osseuse de souris. Caracterisation des echanges membranaires entre la surface cellulaire et les differents compartiments vacuolaires (pinosomes, endosomes, lysosomes) au cours de la pinocytose chez des macrophages non infectes ou infectes par une bacterie non pathogene (bacillus subtilis) ou pathogene (m. Avium)
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ALFRED, VICTOR IFEOLUWA. "GENETIC SCREENING TO IDENTIFY INTERACTORS OF ESCRT-II SUBUNIT, VPS25, AND PRELIMINARY CHARACTERISATION OF CANDIDATES." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/560382.
Full textAbstract:
ESCRT (Endosomal Sorting Complex Required for Transport) proteins regulate cell surface receptor degradation by sorting and packaging ubiquitinated cargoes into the intraluminal vesicles of multivesicular bodies (MVBs). A range of human diseases including cancer, and neurodegeneration display altered expression or are caused by mutations of ESCRT subunits. Studies have shown that Drosophila tissues lacking ESCRTs display neoplastic-like features like overproliferation and polarity defects, partly due to aberrant signalling including Notch signalling. To understand ESCRT-regulated processes in vivo, we utilised modification of a deformed wing phenotype specifically caused by knockdown (RNAi) of Vps25, an ESCRT-II subunit. We systematically screened chromosomal regions and identified 204 genetic interactors of Vps25 that enhanced/suppressed the phenotype. They include genes that function in trafficking, signalling, transcription, ion transport and many other biological processes; suggesting that ESCRTs influence a wide range of biological processes. We have focused on a subset of these hits that regulate tissue growth with a secondary screen based on modification of a Delta-driven eye overgrowth phenotype, isolating a subset of 43 genes involved in regulating tissue growth, some of which are novel and uncharacterised. Human orthologues of some of these genes are important in cancers; dropout (dop), whose mammalian orthologues are the MAST kinases, have been shown to contribute towards breast cancer development. dop mediates Delta-driven eye overgrowth possibly by upregulating Delta expression. In human cells, MAST2 does not affect Notch signalling but might contribute to tumorigenesis by regulating the NFκB pathway. We have also characterised another interactor, CG12163 which is the homologue of mammalian Cathepsin F. Mutations in Cathepsin F cause a rare form of neuronal ceroid lipofuscinosis (NCL) called Type B Kufs disease. Our Drosophila model which recapitulates aspects of the human disease phenotype suggests that defects in autophagy might underlie the pathogenesis of NCLs.
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Parent, Nicolas. "Mécanismes d'activation de la voie lysosomale durant l'apoptose chimio-induite." Thèse, 2009. http://hdl.handle.net/1866/4146.
Full textAbstract:
L’apoptose est une forme de mort cellulaire essentielle au développement et au
maintien de l’homéostase chez les animaux multicellulaires. La machinerie apoptotiq ue
requiert la participation des caspases, des protéases conservées dans l’évolution et celle des
organelles cytoplasmiques. Les lysosomes subissent des ruptures partielles, labilisation de
la membrane lysosomale (LML), qui entraînent l’activation des cathepsines dans le
cytoplasme de cellules cancéreuses humaines en apoptose induite par la camptothecin
(CPT), incluant les histiocytes humains U-937. Ces modifications lysosomales se
manifestent tôt durant l’activation de l’apoptose, concomitamment avec la perméabilisation
de la mitochondrie et l’activation des caspases.
Une étude protéomique quantitative et comparative a permis d’identifier des
changements précoces dans l’expression/localisation de protéines lysosomales de cellules
U-937 en apoptose. Lors de deux expériences indépendantes, sur plus de 538 protéines
lysosomales identifiées et quantifiées grâce au marquage isobarique iTRAQ et LC-ESIMS/
MS, 18 protéines augmentent et 9 diminuent dans les lysosomes purifiés de cellules en
cours d’apoptose comparativement aux cellules contrôles. Les candidats validés par
immuno-buvardage et microscopie confocale incluent le stérol-4-alpha-carboxylate 3-
déhydrogénase, le prosaposin et la protéine kinase C delta (PKC-d). Des expériences
fonctionnelles ont démontrées que la translocation de PKC-d aux lysosomes est requise
pour la LML puisque la réduction de son expression par ARN interférents ou l’inhibition de
son activité à l’aide du rottlerin empêche la LML lors de l’apoptose induite par la CPT. La
translocation de PKC-d aux lysosomes conduit à la phosphorylation et l’activation de la
sphingomyelinase acide lysosomale (ASM), et à l’accroissement subséquent du contenu en
céramide (CER) à la membrane lysosomale. Cette accumulation de CER endogène aux
lysosomes est un évènement critique pour la LML induite par la CPT car l’inhibition de
l’activité de PKC-d ou de ASM diminue la formation de CER et la LML.Ces résultats révèlent un nouveau mécanisme par lequel la PKC-d active
l’ASM qui conduit à son tour à l’accumulation de CER à la membrane lysosomale et
déclenche la LML et l’activation de la voie lysosomale de l’apoptose induite par la CPT. En somme, ce mécanisme confirme l’importance du métabolisme des sphingolipides dans
l’activation de la voie lysosomale de l’apoptose.Apoptosis is a distinct form of regulated cell death which is essential for the development and homeostasis maintenance of multicellular animals. Apoptosis is an evolutionary conserved process involving a specific molecular pathway, known as the caspase cascade, and the different cytoplasmic organelles. A lysosomal pathway, characterized by partial rupture, labilization of lysosomal membranes (LML), and cathepsin activation in the cytoplasm, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In two independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labelling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-d). Functional experiments demonstrate that PKC-d translocation to lysosomes is required for LML, as silencing its expression with RNA interference or suppressing its activity with the inhibitor rottlerin prevents CPT-induced LLM. PKC-d translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase of ceramide (CER) content at lysosomes. The accumulation of endogenous CER at lysosomes is a critical event for CPT-induced LLM as suppressing PKC-d or ASM activity reduces both CPT-mediated CER generation at lysosomes and CPT-induced LLM.These findings reveal a novel mechanism by which PKC-d mediates ASM phosphorylation/activation and CER accumulation at lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. Taken together, these results confirm the importance of sphingolipid metabolism in the activation of the lysosomal pathway of apoptosis.
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Schieweck, Oliver. "Proteomanalyse lysosomaler Membranen: Identifizierung und Charakterisierung neuer lysosomaler Membranproteine." Doctoral thesis, 2008. http://hdl.handle.net/11858/00-1735-0000-0006-B66F-D.
Full text
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Schieweck, Oliver [Verfasser]. "Proteomanalyse lysosomaler Membranen : Identifizierung und Charakterisierung neuer lysosomaler Membranproteine / vorgelegt von Oliver Schieweck." 2008. http://d-nb.info/995573980/34.
Full text
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Hung, Cheng-Yen, and 洪承延. "Purification and Characterization of Porcine Liver Lysosomal Membrane Sialidase." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/50787697478939002358.
Full textAbstract:
碩士國立陽明大學
生物化學研究所
88
Sialidase (nanH) is a group of glycohydrolytic enzyme, widely distributed in nature, that cleaves sialic acid residues from the oligosaccharide components of glycoconjugates. In this study, the porcine liver sialidase was purified by Fractogel TSK CM650 gel, DEAE Sephacel, p-aminophenyloxamic acid agarose, and heparin agarose chromatographies. We obtained two sialidase preparations, SIAⅠand SIAⅡ, which showed the molecular weight of 55 kDa and 49 kDa, respectively, on the SDS-polyacrylamide gel electrophoresis. By Western blotting, using antibody against the SIA Ⅰ, we found that 49 kDa is a protein truncated from 55 kDa in the purification processes. The two enzyme preparations showed an acidic optimum pH, and hydrolyzed gangliosides not only GM3 and GD1a but also GM1 and GM2, which resemble the characteristics of lysosomal membrane sialidase. By immunohistological staining of porcine liver section observed with transmission electron microscope, we confirmed that the sialidase is localized on the lysosomal membrane. The sialidase of Micromonospora viridifaciens has three domains: active site, galactose-binding domain, and immunoglobulin module. In our purification process, we also found that porcine liver sialidase was bound by protein G column, which suggests that the porcine liver sialdase has an immunoglobulin module similar to that of the bacterial sialidase. We have cloned the full-length cDNA of human liver lysosomal matrix sialidase by RT-PCR for the purpose of expressing the human liver lysosomal matrix sialidase in E. coli . The purified sialidase will be used for studying the 3D structure by X-ray crystallography in the future. 英文摘要 --------------------------- 3 緒論 --------------------------- 4 材料與方法 --------------------------- 11 結果 --------------------------- 22 討論 --------------------------- 27 參考文獻 --------------------------- 32 圖表 --------------------------- 38 附錄 --------------------------- 57
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Richtrová, Eva. "Charakterizace promotorových oblastí genů HGSNAT a GBA, a příspěvek ke studiu patogeneze MPS IIIC a Gaucherovy choroby." Doctoral thesis, 2014. http://www.nusl.cz/ntk/nusl-332280.
Full textAbstract:
Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...
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"Membrane anchor for vacuolar targeting: expression of a human lysosomal enzyme iduronidase (hIDUA) in transgenic tobacco plants." 2005. http://library.cuhk.edu.hk/record=b5896394.
Full textAbstract:
Seto Tai Chi.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 122-138).
Abstracts in English and Chinese.
Thesis Committee --- p.ii
Statement --- p.iii
Acknowledgements --- p.iv
Abstract (in English) --- p.v
Abstract (in Chinese) --- p.vii
Table of Contents --- p.ix
List of Tables --- p.xvi
List of Figures --- p.xv
Chapter Chapter 1 --- General Introduction and Literature Review --- p.1
Chapter 1.1 --- Introduction --- p.2
Chapter 1.2 --- Tobacco seed as bioreactor --- p.4
Chapter 1.2.1 --- Advantages of using tobacco seed to produce bioactive human lysosomal enzyme --- p.4
Chapter 1.2.2 --- Disadvantages and potential problems of using tobacco seed to produce bioactive human lysosomal enzyme --- p.5
Chapter 1.2.2.1 --- Difference of asparagine-linked N-glycosylation between plant and human protein --- p.8
Chapter 1.2.2.2 --- Immunogenicity of recombinant protein with plant-derived N-glycan to human --- p.10
Chapter 1.2.2.3 --- "Strategy to ""humanize"" plant-derived recombinant human lysosomal enzyme" --- p.10
Chapter 1.2.2.4 --- Lack of specific glycan structure一mannose-6-phosphate (M6P) tag addition --- p.11
Chapter 1.2.2.5 --- Strategy for M6P tag addition on plant-derived human lysosomal enzyme --- p.12
Chapter 1.3 --- The plant secretory pathway --- p.13
Chapter 1.3.1 --- Plant vacuole in tobacco seed --- p.16
Chapter 1.3.2 --- Soluble protein trafficking in plant cell --- p.17
Chapter 1.3.3 --- Integral membrane protein trafficking in plant cell --- p.17
Chapter 1.3.4 --- Components involved in integral membrane protein trafficking to PSV crystalloid --- p.19
Chapter 1.3.4.1 --- BP-80 (80-kDa binding protein) --- p.19
Chapter 1.3.4.2 --- α-TIP (α-tonoplast intrinsic protein) --- p.20
Chapter 1.3.5 --- Using specific integral membrane protein trafficking system to target recombinant human lysosomal enzyme to tobacco seed PSV --- p.21
Chapter 1.4 --- Homo sapiens α-L-iduronidase (hIDUA) --- p.21
Chapter 1.4.1 --- Global situation of lysosomal storage disease一hIDUA deficiency --- p.21
Chapter 1.4.2 --- Physiological role --- p.22
Chapter 1.4.3 --- Molecular property --- p.24
Chapter 1.4.3.1 --- Mutation and polymorphism --- p.24
Chapter 1.4.4 --- Lysosomal secretory pathway --- p.24
Chapter 1.4.5 --- Biochemical property --- p.25
Chapter 1.4.6 --- Clinical application --- p.27
Chapter 1.4.6.1 --- Enzyme replacement therapy (ERT) --- p.27
Chapter 1.4.6.2 --- Clinical trial --- p.28
Chapter 1.4.6.3 --- Economic value --- p.29
Chapter 1.4.7 --- Expression system --- p.29
Chapter 1.4.7.1 --- Production (overexpression) of rhIDUA in CHO cell system --- p.30
Chapter 1.4.7.2 --- Production of rhIDUA in tobacco plant leaf --- p.30
Chapter 1.5 --- Project objective and long-term significance --- p.30
Chapter 1.5.1 --- Project objective --- p.30
Chapter 1.5.2 --- Long-term significance --- p.31
Chapter Chapter 2 --- Generation and Characterization of Anti-IDUA Antibodies --- p.32
Chapter 2.1 --- Introduction --- p.33
Chapter 2.2 --- Materials --- p.33
Chapter 2.2.1 --- Chemical --- p.33
Chapter 2.3 --- Methods --- p.35
Chapter 2.3.1 --- Generation of polyclonal anti-IDUA antibody --- p.35
Chapter 2.3.1.1 --- Design of synthetic peptide --- p.35
Chapter 2.3.1.2 --- Conjugation of synthetic peptide to carrier protein --- p.39
Chapter 2.3.1.3 --- Immunization of rabbit --- p.39
Chapter 2.3.2 --- Characterization of polyclonal anti-IDUA antibody in rabbit serum --- p.40
Chapter 2.3.2.1 --- Dot-blot analysis --- p.40
Chapter 2.3.3 --- Purification of polyclonal anti-IDUA antibody --- p.42
Chapter 2.3.3.1 --- Construction of anti-IDUA antibody affinity column --- p.42
Chapter 2.3.3.2 --- Affinity-purification of anti-IDUA antibody --- p.42
Chapter 2.3.4 --- Western blot detection of denatured rhIDUA --- p.42
Chapter 2.4 --- Results --- p.43
Chapter 2.4.1 --- Characterization of polyclonal anti-IDUA antibody --- p.43
Chapter 2.5 --- Discussion --- p.51
Chapter 2.6 --- Conclusion --- p.51
Chapter Chapter 3 --- Generation and Characterization of Transgenic Tobacco Plants Expressing rhIDUA Fusions --- p.52
Chapter 3.1 --- Introduction --- p.53
Chapter 3.1.1 --- Signal peptide of hIDUA (hIDUA SP) --- p.54
Chapter 3.1.2 --- Signal peptide of proaleurain (Pro. SP) --- p.54
Chapter 3.1.3 --- Hypothesis to be tested in this study --- p.54
Chapter 3.2 --- Materials --- p.55
Chapter 3.2.1 --- Chemical --- p.55
Chapter 3.2.2 --- Primers --- p.55
Chapter 3.2.3 --- Bacterial strain --- p.58
Chapter 3.2.4 --- The insert-Homo sapiens α-L-iduronidase (hIDUA) cDNA used in this study --- p.58
Chapter 3.2.5 --- The vector-pLJ526 used in this study --- p.59
Chapter 3.3 --- Methods --- p.61
Chapter 3.3.1 --- Construction of chimeric gene construct --- p.61
Chapter 3.3.1.1 --- Restriction endonuclease´ؤPfIMIl --- p.61
Chapter 3.3.1.2 --- Recombinant DNA and molecular cloning techniques used in this study --- p.61
Chapter 3.3.1.3 --- Cloning of pSPIDUA-FLAG --- p.62
Chapter 3.3.1.4 --- Cloning of pSPIDUA-control --- p.62
Chapter 3.3.1.5 --- Cloning of a universal construct (pUniversal) --- p.62
Chapter 3.3.1.6 --- Cloning of pSP-IDUA-T7 --- p.66
Chapter 3.3.1.7 --- Cloning of pSP-IDUA-control --- p.66
Chapter 3.3.1.8 --- Cloning of chimeric gene construct into Agrobacterium binary vector --- p.66
Chapter 3.3.2 --- Expression of chimeric gene construct in tobacco plant --- p.73
Chapter 3.3.2.1 --- Tobacco plant --- p.73
Chapter 3.3.2.2 --- Electroporation of Agrobacterium --- p.73
Chapter 3.3.2.3 --- Agrobacterium-mediated transformation of tobacco plant --- p.74
Chapter 3.3.2.4 --- Selection and regeneration of tobacco transformant --- p.75
Chapter 3.3.3 --- Characterization of transgenic tobacco plant expressing rhIDUA fusion --- p.75
Chapter 3.3.3.1 --- Genomic DNA polymerase chain reaction (PCR) --- p.75
Chapter 3.3.3.2 --- Southern blot analysis --- p.76
Chapter 3.3.3.3 --- Total RNA reverse transcription-PCR (RT-PCR) --- p.77
Chapter 3.3.3.4 --- Northern blot analysis of tobacco leaf --- p.78
Chapter 3.3.3.5 --- Western blot analysis --- p.79
Chapter 3.3.4 --- Purification of plant-derived rhIDUA fusion --- p.81
Chapter 3.3.4.1 --- Construction of affinity column with anti-IDUA antibody --- p.81
Chapter 3.3.4.2 --- Affinity-purification of rhIDUA fusion from tobacco mature seed --- p.81
Chapter 3.3.5 --- Confocal immunoflorescence study --- p.82
Chapter 3.3.5.1 --- Preparation of paraffin section --- p.82
Chapter 3.3.5.2 --- Single immunocytochemical labeling --- p.82
Chapter 3.3.5.3 --- Double labeling with one monoclonal and one polyclonal antibodies --- p.83
Chapter 3.3.5.4 --- Double labeling with two polyclonal antibodies --- p.83
Chapter 3.3.5.5 --- Image collection --- p.84
Chapter 3.4 --- Results --- p.85
Chapter 3.4.1 --- Chimeric gene construction and confirmation --- p.85
Chapter 3.4.2 --- Selection and regeneration of tobacco transformant with kanamycin- resistance --- p.86
Chapter 3.4.3 --- Genomic DNA PCR screening of tobacco transformant --- p.88
Chapter 3.4.4 --- Southern blot analysis of tobacco transformant --- p.91
Chapter 3.4.5 --- Total RNA RT-PCR screening of tobacco transformant --- p.93
Chapter 3.4.6 --- Northern blot analysis of tobacco transformant --- p.93
Chapter 3.4.7 --- Western blot analysis --- p.96
Chapter 3.4.7.1 --- Western blot analysis of pSP-IDUA-T7-121 transformant leaf --- p.96
Chapter 3.4.7.2 --- Western blot analysis of pSP-IDUA-T7-121 transformant mature seed --- p.98
Chapter 3.4.8 --- Affinity-purification of rhIDUA fusion --- p.98
Chapter 3.4.9 --- Expression level of rhIDUA fusion --- p.102
Chapter 3.4.10 --- Subcellular localization of rhIDUA fusion --- p.102
Chapter 3.5 --- Discussion --- p.111
Chapter Chapter 4 --- Summary and Future Perspectives --- p.117
References --- p.122
Appendix 1 --- p.139
Appendix II (List of Abbreviations) --- p.141
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Cristóvão, Beatriz Isabel Pelicano. "Lysosomal function during candida infection: connexin 43 as a new player in the infection process." Master's thesis, 2021. http://hdl.handle.net/10316/98780.
Full textAbstract:
Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de MedicinaInvasive Candidiasis (IC), a life-threatening fungal infection, can be caused by several Candida spp., being Candida albicans the most prevalent specie worldwide. C. albicans is a constituent of the microbiome in most healthy individuals. However, upon perturbations of host immunity, this fungus transits to an opportunistic pathogen. Macrophages are part of the first line of defense against infections as they can engulf and eliminate C. albicans through phagocytosis. After C. albicans engulfment, the yeast transits to a hyphal form inducing phagosomal membrane damage. In order to survive, macrophages require an efficient cellular response to rapidly counteract the hypha-induced cellular damage. Recently, the maintenance of the phagosomal membrane integrity was positively related with C. albicans length growth and escape. Furthermore, a new defense mechanism involving actin-contractile rings structures was elegantly described during C. albicans infection. Thus, considering these emerging studies and the lack of knowledge on the underlying molecular mechanisms involved in hypha-containing phagolysosomal membrane repair, we aimed to investigate the crucial mechanisms responsible to maintain phagolysosomal membrane integrity in macrophages infected with C. albicans by using a RAW 264.7 macrophage cell line. Our results suggested a potential activation of the repair machinery, through the recruitment of Alix and Tsg101, both components of the endosomal sorting complex required for transport (ESCRT) machinery, to C. albicans containing-phagosomes. In addition, we also observed a recruitment of Trim16, a lysophagy component, to the C. albicans-containing phagosome. Moreover, our results indicated that lysosomal biogenesis and autophagy were not the main defence mechanisms involved in macrophages response to C. albicans infection. Notably, we demonstrated that macrophage folding capacity decreased with loss of phagosomal membrane integrity and inhibition of autophagy. Lastly, to unveil new putative players involved in macrophages response to expanding hypha, we also performed an analysis of published transcriptomic data from C. albicans infected macrophages. Our findings highlighted Cx43 as a potential hit during C. albicans infection. Indeed, we found that Cx43 is recruited to phagosomes containing C. albicans and potentiate the folding capacity of macrophages. We also observed a high colocalization of Cx43 with the actin ring structures surrounding the C. albicans-containing phagosome. Taken together, our data unveiled new insights on the impact of the phagosomal membrane integrity, autophagy and lysosomal biogenesis, as well as a new possible non-canonical role of Cx43 on the macrophage immune response to C. albicans infection.
Candidíase Invasiva (CI), uma infeção fúngica grave, pode ser causada por diversas espécies do género Candida, sendo Candida albicans a espécie mais predominante a nível mundial. C. albicans faz parte da flora da maior parte dos indivíduos saudáveis. Porém quando a imunidade do hospedeiro sofre perturbações, este fungo transita para um microrganismo oportunista. Os macrófagos fazem parte da primeira linha de defesa, tendo estas células a capacidade de internalizar e eliminar C. albicans, através da fagocitose. Após a internalização da C. albicans, o fungo sofre alterações na sua morfogénese, transitando para a forma de hifa, induzindo dano na membrana do fagossoma. Para garantir a sua sobrevivência, os macrófagos necessitam de uma resposta celular eficiente para combater rapidamente os danos celulares provocados pela hifa. Recentemente, a manutenção da integridade da membrana do fagossoma foi positivamente relacionada com o crescimento do tamanho da C. albicans e com a sua fuga. Foi também descrito um novo mecanismo de defesa envolvendo estruturas contráteis de actina em forma de anel, durante a infeção por C. albicans. Considerando estes estudos recentes, assim como a falta de conhecimento sobre os mecanismos moleculares envolvidos na reparação da membrana do fagossoma contendo a hifa, o nosso estudo teve como objetivo investigar os principais mecanismos responsáveis pela manutenção da integridade da membrana do fagossoma, utilizando uma linha de macrófagos RAW 264.7 infetados com C. albicans. Os nossos resultados sugeriram uma possível ativação da maquinaria de reparação, através do recrutamento da Alix e da Tsg101, ambas componentes da maquinaria de reparação endosomal sorting complex required for transport (ESCRT), para os fagossomas contendo C. albicans. Adicionalmente, observámos também o recrutamento de Trim16, um componente da maquinaria envolvida na lisofagia, para fagossomas contendo C. albicans. Os nossos resultados também indicaram que os mecanismos de biogénese lisossomal e de autofagia não fazem parte dos principais mecanismos de defesa envolvidos na resposta do macrófago à infeção por C. albicans. Adicionalmente demonstrámos que a capacidade do macrófago de dobrar hifas diminui com a perda da integridade da membrana do fagossoma e com a inibição da autofagia. Por último, para revelar novos intervenientes na resposta do macrófago à hifa em crescimento, realizámos também uma análise de dados publicados de transcriptómica de macrófagos infetados com C. albicans. Os nossos resultados demonstraram a conexina43 (Cx43) como um potencial hit durante a infeção por C. albicans. Efetivamente, foi observado um aumento do recrutamento da Cx43 para o fagossoma contendo C. albicans, assim como um aumento da capacidade dos macrófagos de dobrar as hifas. Encontrámos também uma grande colocalização de Cx43 com as estruturas de actina que se encontravam ao redor do fagossoma contendo C. albicans. Em suma, o nosso trabalho revela novos dados sobre o impacto da integridade da membrana do fagossoma, da autofagia e da biogénese lisossomal, assim como um potencial papel novo não canónico da Cx43 na resposta imune do macrófago à infeção por C. albicans.
Outro - This work was financed by the European Regional Development Fund (ERDF), through the Operational Program for Competitiveness Factors (COMPETE; under the projects PAC “NETDIAMOND” POCI-01-0145-FEDER-016385; HealthyAging2020 CENTRO-01- 0145-FEDER-000012-N2323; POCI-01-0145-FEDER-007440, CENTRO-01-0145- FEDER-032179, CENTRO-01-0145-FEDER-032414, POCI-01-0145-FEDER-022122; FCTUID/NEU/04539/2013, UID/NEU/04539/2019, UIDP/04539/2020; and FCT/PT2020-02/SAICT/2017_424 Repair
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Chang, Melissa Han Yin 1977. "Characterisation and functional studies of the Lysosome-associated membrane protein (LAMP-1) in circulation / by Melissa Chang Han Yin." Thesis, 2003. http://hdl.handle.net/2440/21961.
Full textAbstract:
"February, 2003"Addendum inside front cover.
Bibliograpy: leaves 200-219.
xiv, 219, xiv leaves : ill. (some col.), plates (some col.) ; 30 cm.
Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003
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Chang, Melissa Han Yin 1977. "Characterisation and functional studies of the Lysosome-associated membrane protein (LAMP-1) in circulation / by Melissa Chang Han Yin." 2003. http://hdl.handle.net/2440/21961.
Full textAbstract:
"February, 2003"Addendum inside front cover.
Bibliograpy: leaves 200-219.
xiv, 219, xiv leaves : ill. (some col.), plates (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003
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Bodammer, Beatrice. "Untersuchung der Acetyl-Coenzym A:alpha-Glucosaminid N-Acetyltransferase in gereinigten lysosomalen Membranen." Doctoral thesis, 2012. http://hdl.handle.net/11858/00-1735-0000-000D-EF66-A.
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ZHANG, HUI-ZHEN, and 張慧珍. "Localization of porcine spleen acid deoxyribonuclease in lysosmes and reassociation of the enzyme with the lysosomal membrane." Thesis, 1987. http://ndltd.ncl.edu.tw/handle/80550301321790072392.
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Huynh, Kassidy. "Phagosome Maturation: Aging with pH, Lysosome-associated membrane proteins, and Cholesterol; while staying young with Burkholderia cenocepacia." Thesis, 2009. http://hdl.handle.net/1807/19278.
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Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial
remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many
protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while
ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.
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Savalas, Lalu Rudyat Telly [Verfasser]. "Biochemical characterization of a novel lysosomal membrane protein disrupted in renal carcinoma 2 (DIRC2) / submitted by Lalu Rudyat Telly Savalas." 2011. http://d-nb.info/1012739732/34.
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