Background and objectives: Growing bodies of evidence demonstrate that adipocyte fatty acid binding protein (A-FABP) mediates the pathogenesis of atherosclerosis through its direct impacts on macrophages. Loss-of-function study was conducted by utilizing peritoneal macrophages derived from A-FABP knockout (KO) mice, to investigate the role of A-FABP in autophagy and macrophage foam cell formation. Key findings: 1. No morphological changes between the peritoneal macrophages derived from A-FABP knockout (KO) or their wild-type (WT) littermates. 2. Foam cell formation was successfully induced by the treatment of acetylated low-density lipoproteins (LDL) in peritoneal macrophages derived from A-FABP WT and KO mice. 3. LDL treatment induces autophagy in peritoneal macrophages from both A-FABP WT and KO mice. 4. The extent of LDL-induced autophagy is reduced in peritoneal macrophages of WT mice and is accompanied by increased lipid droplet accumulation when compared with A-FABP KO mice. Conclusions: A-FABP is a suppressor of autophagy and contributes to the attenuation of cholesterol efflux, subsequently resulting in enhancement of lipid droplets accumulation in peritoneal macrophages. A-FABP mediates the formation of macrophage foam cell via the suppression of autophagy. The results suggest that A-FABP is a potential therapeutic target to suspend the progression of atherosclerosis and remit the atherosclerotic lesion. / published_or_final_version / Medicine / Master / Master of Medical Sciences

No description available.

Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T08:32:23Z (GMT). No. of bitstreams: 1 Oliveira_Luciana_M.pdf: 438742 bytes, checksum: a5f9e3b963ae7457238c742a983b8733 (MD5) Previous issue date: 2013 / Resumo: Aeromonas são bacilos Gram-negativos, anaeróbios facultativos, ermentadores, oxidase e catalase positivos, ubíquos em ambientes aquáticos e capazes de causar uma variedade de doenças em humanos, principalmente gastroenterite. As espécies mais comumente associadas com infecções em humanos são A. hydrophila, A.caviae e A. veronii biovar sobria. Entre os vários fatores de virulência descritos, Aerolisina é considerada um dos principais fatores de virulência do genero Aeromonas, sendo esta além de provocar a lise em eritrócitos de carneiro tem atividade enterotóxica. Recentemente foi descrito pelo nosso uma enterotoxina citotóxica denominada Fator Citotóxico Vacuolizante (VCF), diferente de Aerolisina biologica e molecularmente, no sobrenadante da cultura de Aeromonas veronni biovar sobria, isoladas de casos clinicos. O VCF purificado apresentou atividade citotóxica em linhagens de células tumorais humanas tais como, HEp-2, Caco-2, HeLa e NCI-H 292. No ensaio de efeito anti-proliferativo o VCF apresentou potencial atividade de inibição de crescimento de células tumorais NCI-ADR/RES, uma linhagem celular com resistência a múltiplas drogas de quimioterapia e K562, uma linhagem de células leucêmicas. Estes resultados sugerem que o VCF apresenta potencial toxigênico sobre células tumorais de origem humana / Abstract: Aeromonas is a Gram-negative, facultative anaerobic rod, fermenting, oxidase and catalase positive ubiquitous in fresh and brackish water and capable of causing several human diseases, specially gastroenteritis. The most important pathogens species associated with human infections are A. hydrophila, A. caviae, and A. veronii biovar sobria. Between the several virulence factors described, Aerolisin is considered as one of the main virulence factors of the Aeromonas genus, this one causing lysis in lamb erythrocytes and has an enterotoxic activity. Was recently described by our a citotoxic enterotoxin named as Vacuolating Cytotoxic Factor (VCF), differing biologicaly and molecularly from the Aerolisin, at the supernatant of a culture of A. veronii biovar sobria, isolated from clinic cases. The purified VCF presented citotoxic activitiy on lineages of human tumorous cells such as , HEp-2, Caco-2, HeLa and NCI-H 292. On the test antiproliferative effect the VCF presented a potencial activity of growth inhibition on tumour cells NCI-ADR/RES, a cellular lineage with resistance to multiple chemotherapy drugs and K562, a lineage of leukemic cells. This results sugests that the VCF presents toxigenic potencial over human tumorous cells / Mestrado / Clinica Medica / Mestra em Clínica Médica

Cytoplasmic dynein is a highly conserved multi-subunit motor protein that transports a variety of cellular cargoes, including proteins and organelles, towards minus ends of microtubules. Dynein is recruited to specific subclasses of cellular organelles via a specialized class of adaptor proteins, that serve as physical scaffolds for dynein recruitment to cargoes. Recent work shows that these adaptor proteins are also capable of altering biophysical properties of dynein in vitro and in vivo. This work now finds that a dynein adaptor protein, RILP, through multiple interactors, coordinates the progression of a complex biological pathway. Autophagy is a multi-step, highly conserved pathway that involves de novo formation of a double-membraned autophagosome around ubiquitinated cellular cargoes including long-lived proteins and damaged organelles for subsequent degradation by the lysosome. My work finds a dynein adaptor protein, RILP, to control not only retrograde microtubule-based autophagosome transport but their formation as well. RILP achieves these functions by sequentially interacting with the isolation membrane protein, ATG5, and the autophagosome membrane protein, LC3. During autophagosome formation, ATG5 competes with dynein to bind to a common site within the RILP N-terminus to prevent premature initiation of autophagosome motility. Depletion or LC3-interacting site mutations in RILP prevent formation of autophagosomes as well as impede their retrograde transport. This in turn results in an accumulation of ubiquitinated cargoes, including p62/ Sequestosome-1 in cells, showing that RILP is essential for autophagic clearance in cells, a finding that has broad implications for aggregate-prone neurodegenerative diseases. Finally, this work characterizes the molecular composition of the RILP-dynein supercomplex, and identifies Lis1 (implicated in lissencephaly) as an obligate component of the RILP supercomplex. Interestingly, another dynein regulator, NudE (implicated in microcephaly) is absent. Lis1 depletion results in RILP vesicle dispersion, suggesting that it is needed for RILP-mediated dynein driven transport. Altogether, these findings show for the first time that dynein adaptor RILP controls a complex multi-step biological pathway. The unique composition of RILP supercomplex holds new possibilities for dynein regulation in vivo.

Macroautophagy is a degradative system that cells employ to degrade proteins, lipids, pathogens or whole organelles. Dysfunctional autophagy has been implicated in diseases ranging from cancer to neurodegeneration. Animal models lacking macroautophagy fail to preserve a functional liver or central nervous system, supporting the importance of autophagy in maintaining the health of these tissues. However, it is unclear why this degradative pathway is critical in maintaining homeostasis. All macroautophagic cargo are sequestered by the multilamellar organelle called the autophagosome. The formation of the autophagosome depends on the lipidation of a cytosolic protein LC3, so that it associates with the autophagosomal membrane throughout the autophagic process. Using a mouse model expressing GFP-LC3, we have developed an approach to immunopurify autophagosomes from different tissue, then identified their autophagosomal content using tandem-mass-tag (TMT) quantitative proteomics. We have found that the tissues rely on autophagy differently based on the turnover of their organelles as liver depended more on autophagy for ER turnover and brain relied on autophagy more for mitochondrial turnover and its synaptic vesicle homeostasis. Starvation can activate macroautophagy, and is the most studied means through which this pathway has been studied. The importance of autophagy activation in the liver during starvation has been well characterized whereas its importance in the brain has been debated. In this study, we have found that both the liver and brain rely on autophagic degradation of mitochondria differently during starvation. As expected, liver increases its autophagic response upon 24 hr nutrient deprivation, but surprisingly cargo capture transitions from whole mitochondrial turnover to piecemeal mitochondrial turnover. In contrast, in brain, mitochondria-turnover remains largely unchanged. Moreover, although neuronal cargo proteins also remained largely unchanged in response to nutrient deprivation, there was a robust response driven by the non-neuronal cells of the CNS including glial cells and brain endothelial cells, indicating how the discrete cell types of the CNS respond to this physiologic stressor differently. Taken together, this work reveals the tissue-specificity and cell-specificity in the physiological role of autophagy, providing insight in how vertebrates use autophagy to maintain health and react to stress.

This study investigated the morphologic differences of the giant vacuoles (GVs) in the inner wall endothelium of Schlemm’s canal (SC) in human eyes perfused at either 30 mmHg or 7 mmHg (physiologic pressure in enucleated eyes) using serial block-face scanning electron microscopy (SBF-SEM) paired with three-dimensional reconstruction software. Two normal human eyes were perfused at 30 mmHg with fluorescent tracers to mark regions of active and inactive flow, followed by perfusion-fixation. Tissue wedges (n = 6) of trabecular meshwork including SC from high-, low-, and non-flow areas of each eye (determined by tracer distribution) were dissected and processed for SBF-SEM. Four types of GVs were identified: Type I GVs which lack both a basal opening and an Ipore; Type II GVs which have a basal opening but lack an I-pore; Type III GVs which have an I-pore but lack a basal opening; and Type IV GVs which possess both a basal opening and an I-pore. Types and spans of GVs were collected from the SBF-SEM images, and volumes of GVs from a random subset were measured using 3D reconstruction. Results were compared with findings from an earlier study conducted with two eyes perfused at 7 mmHg and prepared in the same manner (Soares, 2022). In total, 19,047 SBF-SEM images were analyzed between 7 mmHg (n = 9586) and 30 mmHg (n = 9461) using Reconstruct. Statistical analysis comparing data between the two pressures was performed using R. There were more GVs found at 30 mmHg (n = 1541) when compared with 7 mmHg (n = 1312), and there were more Type IV GVs at 30 mmHg when compared with 7 mmHg. Type IV GVs occurred most frequently in high-flow areas at both pressures. GVs with I-pores were greater in size (both span and volume) than GVs without I-pores in all flow areas at both pressures. Type IV GVs were larger than Type II GVs which were larger than Type I GVs at both pressures. The span of GVs without I-pores was significantly greater at 7 mmHg. However, there was no significant difference between the volumes of GVs with or without I-pores between the two pressures. The result that GVs with I-pores were larger in size than GVs without I-pores at all conditions appears to support the theory that GV size is an important contributing factor to I-pore formation. The differences in span but not volume of GVs without I-pores between two pressures suggest that GVs at high pressure may be more convex in shape and may protrude further into SC, a situation which could contribute to thinning of the cellular membrane of GVs. Finally, the result that more Type IV GVs were found in high-flow areas at both pressures implies that the changing percentage of Type IV GVs likely plays a role in regulating segmental flow. / 2025-02-26T00:00:00Z

La cardosine A est une protéase aspartique identifiée il y a plus de 20 ans dans les cellules du chardon Cynara cardunculus. Sa distribution dans tous les tissus de la plante et ses caractéristiques enzymatiques ont été caractérisées par approches biochimiques. La cardosine A a des fonctions essentielles dans la reproduction, la mobilisation de réserves protéiques, et le remaniement de membranes. Pour assumer ces différentes fonctions, la cardosine A doit pouvoir transiter et s’accumuler dans différents compartiments intracellulaires : vacuole de stockage, vacuoles lytiques, ou autres compartiments membranaires. Il n’y a cependant que très peu de données disponibles sur les mécanismes de biosynthèse, de tri, de transport et d’adressage aux différents compartiments cellulaires. De récents travaux suggèrent que l’expression en modèle hétérologue pourrait être utilisée pour une meilleure compréhension de la biologie intracellulaire de la cardosine A. Les résultats de cette étude montrent que l’expression transitoire de la cardosine A dans les feuilles de Nicotiana tabacum est un bon modèle expérimental pour explorer le transport de la cardosine A dans la cellule. En effet dans ce système les mécanismes de maturation et de transport de la protéine à la vacuole sont conservés. De plus, une lignée stable d’Arabidopsis thaliana exprimant la cardosine A sous promoteur inductible s’est également avérée un bon modèle d’étude du transport intracellulaire de la cardosine A. L’utilisation de ces systèmes hétérologues a permis de combiner l’expression de formes mutées de la cardosine A (dans lesquelles des séquences spécifiques ou des acides aminés avaient été tronqués ou modifiés) avec des approches de biochimie et d’imagerie cellulaire pour identifier des signatures moléculaires responsables de l’adressage vacuolaire de la protéine. Nos résultats montrent que la cardosine A a deux déterminants vacuolaires dans sa séquence protéique : le domaine “PSI”, qui définit un déterminant d’adressage vacuolaire original et propre à certaines protéases aspartiques, et un peptide C-terminal appartenant à la classe bien définie des ctVSD. De plus, les résultats montrent que la présence de ces deux déterminants illustre la capacité d’emprunter deux routes distinctes pour atteindre la vacuole : le domaine PSI peut permettre d’attendre la vacuole sans passer par le Golgi, tandis que le domaine C-ter négocie un transport classique Reticulum, Golgi, Prévacuole, Vacuole. Cette capacité à choisir deux routes différentes n’est pas observée pour la cardosine B, autre protéase aspartique du chardon présentant une haute homologie de séquence avec la cardosine A. Pour expliquer cette capacité de la cardosine A à emprunter deux routes vacuolaires différentes, l’hypothèse d’un rôle possible de la glycosylation dans le tri des protéines entre les deux routes vacuolaires est alors étudiée. L’expression de la cardosine A dans les graines en germination d’Arabidopsis thaliana révèle que la protéine peut s’accumuler d’une manière différentielle dans les graines en absence de germination ou pendant la germination, tout au long du système endomembranaire jusqu’à la vacuole de réserve ou dans les vacuoles lytiques en formation. Les expériences de blocage de transport du Reticulum au Golgi n’ont pas permis de conclure d’une manière certaine si les accumulations vacuolaires dérivaient d’un transport pouvant court-circuiter le Golgi comme dans les feuilles de Nicotiana. Au total, la cardosine A constitue une protéine modèle pour étudier les transports vacuolaires chez Nicotiana tabacum and Arabidopsis thaliana, deux systèmes hétérologues qui permettent de développer des méthodes complémentaires pour une exploration fonctionnelle des mécanismes impliqués. Cette étude permet de contribuer à une meilleure connaissance de la biologie des cardosines en particulier, et des protéases aspartiques en général. / The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage vacuoles and lytic vacuoles in the flowers and in protein bodies in seeds of the native plant cardoon. Cardosin A has been first isolated almost two decades ago and has been extensively characterized since, both in terms of distribution within the tissues and of enzyme biochemistry. In the native system, several roles have been addressed to cardosin A in reproduction, mobilization of reserves and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments inside the cell: protein storage vacuoles, lytic vacuoles, cytoplasmic membrane (and eventually outside the cell). However, not much information is available regarding cardosin A biogenesis, sorting or trafficking to the different compartments. Recent studies have approached the expression of cardosin A in Arabidopsis thaliana and Nicotiana tabacum. These preliminary observations were the starting point of a detailed study of cardosin A expression, localisation, sorting and trafficking routes, resourcing to several and very different methods. It has been showed that transient expression of cardosin A in Nicotiana tabacum leaf is a good system to explore cardosin A trafficking inside the cell, as the protein is processed in a similar manner as the control and accumulates in the vacuole. Furthermore, an Arabidopsis thaliana line expressing cardosin A under an inducible promoter was explored to understand cardosin A dynamics in terms of vacuolar accumulation during seed germination events. Similarly to the Nicotiana tabacum one, this system was also validated for cardosin A expression and it allowed to conclude that the protein’s expression did not retrieved any phenotype to the cells or individuals. However, experiments conducted in BY-2 cells revealed to be inconclusive since cardosin A expression in this system is not predictable. The data obtained along this work using several cardosin A mutated forms, lacking specific domains or point-mutated, allowed to determine that cardosin A has two Vacuolar Sorting Determinants in its protein sequence: the PSI, an unconventional sorting determinant, and the C-terminal peptide, a C-terminus sorting determinant by definition. Furthermore, it was also demonstrated that each domain represents a different route to the vacuole: the PSI bypasses the Golgi Apparatus and the C-terminal peptide follows a classic Endoplasmic Reticulum-Golgi Apparatus-Prevacuole route to the vacuole. This difference in the trafficking routes is not observed for cardosin B sorting determinants as both the PSI and C-terminal peptide from cardosin B needs to pass the Golgi Apparatus to reach the vacuole. A putative role for glycosylation in the trafficking routes is further discussed as cardosin A PSI, contrary to cardosin B, is not glycosylated. The production of mutants affecting cardosin A glycosylation sites supported this idea. Moreover, cardosin A expression in germinating Arabidopsis thaliana seeds revealed a differential accumulation in non-germinated and germinated seedlings. Cardosin A was detected along the secretory pathway to the Protein Storage Vacuole in association with the Endoplasmic Reticulum, Golgi Apparatus, Prevacuole and newly formed Lytic Vacuoles. The drug Brefeldin A caused the protein to be retained in the Golgi Apparatus, despite some amount being still detected in the vacuole, not being clear if the Golgi Apparatus bypass observed in Nicotiana tabacum leaves occurs in this system. As a whole, cardosin A confirmed to be a good model to study vacuolar sorting in these two systems that complement each other in terms of approaches available. This study provided good results in order to understand in more detail cardosin A biology in particular and vacuolar trafficking of plant Aspartic Proteinases as a group.

Listeria monocytogenes est une bactérie pathogène intracellulaire facultative responsable d’une pathologie grave, la listériose. Si de très nombreux travaux ont permis de caractériser les mécanismes de virulence de cette bactérie, il existe peu de données sur les mécanismes conduisant au portage asymptomatique de L. monocytogenes dans les hôtes mammifères. L’un de ces mécanismes pourrait être une phase de persistance intracellulaire. Lors d’infections prolongées de cellules épithéliales humaines en culture, comme des hépatocytes et des cellules de trophoblastes, L. monocytogenes change de mode de vie intracellulaire. Après la phase active de dissémination de cellule en cellule, les bactéries arrêtent de polymériser l’actine et se retrouvent piégées dans des vacuoles à simple membrane marquées par la protéine endosomale LAMP1. L’objectif de ma thèse était de caractériser ces « Listeria-Containing Vacuoles » (LisCVs). Nous avons montré que les LisCVs sont des compartiments acides, partiellement-dégradatifs, marquées par la protéase lysosomale cathépsine D. Leur formation coïncide avec la disparition du facteur de polymérisation d’actine ActA de la surface bactérienne et la capture des bactéries cytosoliques dépourvues d’actine par des membranes cellulaires. Dans ces compartiments, les bactéries entrent en croissance ralentie ; une sous-population résiste aux stress et peut survivre au-delà de trois jours d’infection. L’utilisation de la gentamicine lors du protocole d’infection n’est pas responsable de la formation des LisCVs. Cependant, cet antibiotique permet la sélection des bactéries vacuolaires, en inhibant spécifiquement la croissance des bactéries cytosoliques. La formation des LisCVs n’est pas spécifique des souches de laboratoire. Toutefois l’efficacité du phénomène pourrait diverger selon les séquençotypes des souches de L. monocytogenes. Les bactéries vacuolaires ont la capacité de sortir des vacuoles et de retourner vers un état motile et réplicatif, après le passage des cellules infectées. Lorsque l’expression du gène actA reste inactive, comme dans les mutants ∆actA, des formes de Listeria vacuolaires persistent dans les cellules hôtes dans un état viable mais non cultivable (VBNC). Ces formes VBNC peuvent être transmises au cours des divisions des cellules hôtes. L’ensemble de ces résultats révèle une nouvelle phase de persistance dans le processus infectieux intracellulaire de L. monocytogenes lors des infections prolongées de certaines cellules épithéliales. Cette propriété pourrait contribuer au portage asymptomatique de ce pathogène dans les tissus épithéliaux, allonger la période d'incubation de la listériose, et rendre les bactéries tolérantes à l’antibiothérapie. / Listeria monocytogenes is a facultative intracellular pathogenic bacterium responsible for a serious disease, listeriosis. Although much work has been done to characterize the virulence mechanisms of this bacterium, there is little data on the mechanisms leading to the asymptomatic carriage of L. monocytogenes in mammalian hosts. One of these mechanisms could be a phase of intracellular persistence. During prolonged infections of human epithelial cells in culture, such as hepatocytes and trophoblast cells, L. monocytogenes changes its intracellular lifestyle. After the active phase of cell-to-cell spread, the bacteria stop polymerizing actin and become trapped in single-membrane vacuoles labeled with the endosomal protein LAMP1.The aim of my thesis was to characterize these "Listeria-Containing Vacuoles" (LisCVs). We have shown that LisCVs are acidic, partially degradative compartments, labeled by the lysosomal protease cathepsin D. Their formation coincides with the disappearance of actin polymerization factor ActA from the bacterial surface and the capture of actin-free cytosolic bacteria by cell membranes. In these compartments, bacterial growth is slowed; a subpopulation is resistant to stress and can survive beyond three days of infection. The use of gentamicin during the infection protocol is not responsible for the formation of LisCVs. However, this antibiotic allows selection of vacuolar bacteria, by specifically inhibiting the growth of cytosolic bacteria. The formation of LisCVs is not specific to laboratory strains. However, the efficacy of the phenomenon could diverge according to the sequence types of L. monocytogenes strains. Vacuolar bacteria have the ability to exit the vacuoles and return to a motile and replicative state during the subculture of infected cells. When expression of the actA gene remains inactive, as in ΔactA mutants, vacuolar Listeria forms persist in host cells in a viable but non-culturable (VBNC) state. These VBNC forms can be transmitted during host cell divisions. All these results reveal a new phase of persistence in the intracellular infectious process of L. monocytogenes during prolonged infections of a subset of epithelial cells. This property could contribute to asymptomatic carriage of this pathogen in epithelial tissues, extend the incubation period of listeriosis, and make bacteria tolerant to antibiotic therapy.

Miao Yansong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 78-83). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The plant secretory pathway --- p.2 / Chapter 2. --- Two different types of vacuoles in plant cells --- p.2 / Chapter 3. --- Vacuolar sorting receptor (VSR) proteins --- p.3 / Chapter 4. --- BP-80 and prevacuolar compartment --- p.6 / Chapter 5. --- The Arabidopsis VSR proteins --- p.7 / Chapter 6. --- Research objectives --- p.8 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing GFP-AtVSR Fusions --- p.10 / Chapter 1. --- Introduction --- p.11 / Chapter 2. --- Materials and Methods --- p.12 / Chapter 2.1 --- Structure of Golgi marker and PVC marker --- p.12 / Chapter 2.2 --- Construction of GFP-VSR reporters --- p.14 / Chapter 2.3 --- Agrobacterium electroporation --- p.24 / Chapter 2.4 --- Transformation of tobacco BY-2 cells --- p.24 / Chapter 2.5 --- Screening of transgenic BY-2 cells expressing GFP-VSR fusions --- p.25 / Chapter 2.6 --- Chemicals --- p.27 / Chapter 3. --- Result.s --- p.28 / Chapter 3.1 --- Chimeric GFP reporters as tools to study subcellular localization of Arabidopsis vacuolar sorting receptor proteins in transgenic BY-2 cells --- p.28 / Chapter 3.2 --- Establishment of transgenic tobacco (Nicotiana tabacum) BY-2 cell lines stably expressing seven GFP-AtVSR reporters --- p.29 / Chapter 4. --- Conclusion --- p.38 / Chapter Chapter 3 --- Subcellular Localization of the Seven GFP-AtVSR Fusions to Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.39 / Chapter 1. --- Introduction --- p.40 / Chapter 2. --- Materials and Methods --- p.41 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.41 / Chapter 2.2 --- Antibodies for immunolabeling --- p.42 / Chapter 2.3 --- Wortmannin and brefeldin A treatment --- p.42 / Chapter 2.4 --- Electron microscopy of resin-embedded cells --- p.43 / Chapter 3. --- Results --- p.44 / Chapter 3.1 --- Vacuolar sorting receptor proteins in plants --- p.44 / Chapter 3.2 --- PVC localization of GFP-AtVSR fusions in transgenic tobacco BY-2 cells --- p.47 / Chapter 3.3 --- The spacer sequences did not affect PVC localization of GFP-AtVSR7 --- p.56 / Chapter 3.4 --- Wortmannin-induced vacuolated PVCs contained VSRs in tobacco BY-2 cells --- p.58 / Chapter 3.5 --- Wortmannin-induced vacuolation of PVCs is a general response in plant cells --- p.62 / Chapter 4. --- Conclusion --- p.65 / Chapter Chapter 4 --- Discussion and Future Perspectives --- p.66 / Chapter 1. --- The hypothesis in this study --- p.67 / Chapter 2. --- GFP and BY-2 cells --- p.67 / Chapter 3. --- A reporter system to study subcellular localization of VSR proteins in transgenic tobacco BY-2 cells --- p.68 / Chapter 4. --- PVC localization of the seven GFP-AtVSR fusions in transgenic BY-2 cells --- p.69 / Chapter 5. --- VSR spacer sequences did not affect PVC localization of GFP-AtVSR fusions in transgenic tobacco BY-2 cells --- p.71 / Chapter 6. --- PVC localization of GFP-PV72 and GFP-AtVSR 1 fusions in transgenic tobacco BY-2 cells --- p.73 / Chapter 7. --- Wortmannin-induced vacuolation of PVC is a general response in plant cells --- p.75 / Chapter 8. --- Future perspectives --- p.75 / References --- p.78