Tyrimų tikslas – naudojant kvantinius taškus (KT), kaip tinkamų vaistų pernašai biosuderinamų nanodalelių modelį, in vitro nustatyti bendrus tikslingai nemodifikuotų nanodalelių patekimo į ląsteles ir lokalizacijos jose dėsningumus. Spektroskopiniais metodais ištyrus KT patekimo į ląsteles laikinę dinamiką nustatyta, kad neigiamo krūvio apvalkalo KT patekimo į ląsteles procesą galima suskirstyti į tris etapus, tačiau šie kaupimosi etapai skirtingose ląstelių kultūrose yra nevienodos trukmės. Konfokalinės fluorescencijos mikroskopijos metodu ištyrus neigiamo paviršiaus krūvio KT patekimo į ląsteles procesą, pagal viduląstelinį KT pasiskirstymą ir susiformavusių pūslelių tipus, galima suskirstyti į keturias fazes. Naudojant fluorescencijos gyvavimo trukmės vaizdinimo mikroskopijos metodą užregistruotas nuo endosomų brandos priklausantis viduląstelinių vezikulių vidinės struktūros heterogeniškumas. Skirtingos brandos endosomos gali būti apibūdinamos ir vaizdinamos remiantis jose sukauptų KT savitomis fotoliuminescencijos gyvavimo trukmėmis. Skirtingais fizikiniais ir biocheminiais metodais nustatyta, kad difuzijos būdu KT per ląstelės membraną neprasiskverbia, bet į ląsteles patenka endocitozės būdu. Nepadengti baltymais neigiamo paviršiaus krūvio KT į ląsteles patenka tik vienu nuo kaveolino priklausančiu endocitozės keliu. KT, kaip modelinių diagnostinių priemonių, ir antivėžinio preparato bendras panaudojimas in vitro atskleidė, kad KT, nesukeldami toksinio poveikio ląstelių... [toliau žr. visą tekstą] / The overall aim of the study is to identify the general rules for intracellular uptake and localisation of non-targeted nanoparticles by employing colloid quantum dots (QDs) as a model of biocompatible nanoparticle-based drug carriers. The investigation of intracellular uptake of non-targeted negatively charged QDs with fluorescence spectroscopy method revealed three time-related accumulation stages, which are characteristic for all investigated cell lines, but the stages were of different timing for each of the investigated cell line. The confocal fluorescence microscopy imaging showed four intracellular accumulation phases of QDs based on type and localisation of formed vesicles. The fluorescence lifetime imaging microscopy revealed the inner heterogeneity of intracellular vesicles: the endosomes at particular stage of maturity can be identified by different photoluminescence lifetimes of accumulated QDs. QDs do not penetrate plasma membranes through passive diffusion, but enter a cell through endocytosis. Negatively charged QDs without protein corona enter the cells through the single, caveolin-dependent endocytic pathway. Examination of the possibility to combine QDs, as model diagnostic probes, with anticancer agent in vitro revealed, that despite QDs alone had no cytotoxic effects on cells viability, they increased drug resistance. Therefore, this effect needs profound further research before the application of quantum dots in combined diagnosis and therapy in vivo.

The overall aim of the study is to identify the general rules for intracellular uptake and localisation of non-targeted nanoparticles by employing colloid quantum dots (QDs) as a model of biocompatible nanoparticle-based drug carriers. The investigation of intracellular uptake of non-targeted negatively charged QDs with fluorescence spectroscopy method revealed three time-related accumulation stages, which are characteristic for all investigated cell lines, but the stages were of different timing for each of the investigated cell line. The confocal fluorescence microscopy imaging showed four intracellular accumulation phases of QDs based on type and localisation of formed vesicles. The fluorescence lifetime imaging microscopy revealed the inner heterogeneity of intracellular vesicles: the endosomes at particular stage of maturity can be identified by different photoluminescence lifetimes of accumulated QDs. QDs do not penetrate plasma membranes through passive diffusion, but enter a cell through endocytosis. Negatively charged QDs without protein corona enter the cells through the single, caveolin-dependent endocytic pathway. Examination of the possibility to combine QDs, as model diagnostic probes, with anticancer agent in vitro revealed, that despite QDs alone had no cytotoxic effects on cells viability, they increased drug resistance. Therefore, this effect needs profound further research before the application of quantum dots in combined diagnosis and therapy in vivo. / Tyrimų tikslas – naudojant kvantinius taškus (KT), kaip tinkamų vaistų pernašai biosuderinamų nanodalelių modelį, in vitro nustatyti bendrus tikslingai nemodifikuotų nanodalelių patekimo į ląsteles ir lokalizacijos jose dėsningumus. Spektroskopiniais metodais ištyrus KT patekimo į ląsteles laikinę dinamiką nustatyta, kad neigiamo krūvio apvalkalo KT patekimo į ląsteles procesą galima suskirstyti į tris etapus, tačiau šie kaupimosi etapai skirtingose ląstelių kultūrose yra nevienodos trukmės. Konfokalinės fluorescencijos mikroskopijos metodu ištyrus neigiamo paviršiaus krūvio KT patekimo į ląsteles procesą, pagal viduląstelinį KT pasiskirstymą ir susiformavusių pūslelių tipus, galima suskirstyti į keturias fazes. Naudojant fluorescencijos gyvavimo trukmės vaizdinimo mikroskopijos metodą užregistruotas nuo endosomų brandos priklausantis viduląstelinių vezikulių vidinės struktūros heterogeniškumas. Skirtingos brandos endosomos gali būti apibūdinamos ir vaizdinamos remiantis jose sukauptų KT savitomis fotoliuminescencijos gyvavimo trukmėmis. Skirtingais fizikiniais ir biocheminiais metodais nustatyta, kad difuzijos būdu KT per ląstelės membraną neprasiskverbia, bet į ląsteles patenka endocitozės būdu. Nepadengti baltymais neigiamo paviršiaus krūvio KT į ląsteles patenka tik vienu nuo kaveolino priklausančiu endocitozės keliu. KT, kaip modelinių diagnostinių priemonių, ir antivėžinio preparato bendras panaudojimas in vitro atskleidė, kad KT, nesukeldami toksinio poveikio ląstelių... [toliau žr. visą tekstą]

Fcγ receptors (FcγR) which recognize the Fc fraction of IgG play key roles in the modulation of a range of cellular responses as part of the host defense against foreign microbes and antigens. An important function of FcγR is to mediate internalization of soluble IgG-containing immune complexes via endocytosis. The mechanisms of internalization and intracellular transport of FcγR after internalization are less clear. In this thesis, I investigated the trafficking behaviours of human FcγRIIA and FcγRIIB2 upon clustering with immune complexes. In Chapter 3, I demonstrate FcγRIIA, when engaged with multivalent heat aggregated IgG (agIgG), is delivered along with its ligand to lysosomal compartments for degradation, whereas FcγRIIB2 becomes dissociated from the ligand and routed separately into a recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase (SFK) activity or receptor ubiquitylation. Upon co-engagement, these two receptors are sorted independently to distinct final fates after dissociating from their co-clustering ligand. In Chapter 4, I show that while the ubiquitin-conjugating system is required for FcγRIIA-mediated endocytosis, it is not required for FcγRIIB2 endocytosis. FcγRIIB2 internalizes immune complexes at a faster rate than FcγRIIA and accelerates the endocytosis of FcγRIIA upon receptor co-engagement. Taken together, these results reveal fundamental differences in the trafficking behaviour of FcγRIIA and FcγRIIB2 both during the initial induction of endocytosis as well as during subsequent intracellular sorting.

Fcγ receptors (FcγR) which recognize the Fc fraction of IgG play key roles in the modulation of a range of cellular responses as part of the host defense against foreign microbes and antigens. An important function of FcγR is to mediate internalization of soluble IgG-containing immune complexes via endocytosis. The mechanisms of internalization and intracellular transport of FcγR after internalization are less clear. In this thesis, I investigated the trafficking behaviours of human FcγRIIA and FcγRIIB2 upon clustering with immune complexes. In Chapter 3, I demonstrate FcγRIIA, when engaged with multivalent heat aggregated IgG (agIgG), is delivered along with its ligand to lysosomal compartments for degradation, whereas FcγRIIB2 becomes dissociated from the ligand and routed separately into a recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase (SFK) activity or receptor ubiquitylation. Upon co-engagement, these two receptors are sorted independently to distinct final fates after dissociating from their co-clustering ligand. In Chapter 4, I show that while the ubiquitin-conjugating system is required for FcγRIIA-mediated endocytosis, it is not required for FcγRIIB2 endocytosis. FcγRIIB2 internalizes immune complexes at a faster rate than FcγRIIA and accelerates the endocytosis of FcγRIIA upon receptor co-engagement. Taken together, these results reveal fundamental differences in the trafficking behaviour of FcγRIIA and FcγRIIB2 both during the initial induction of endocytosis as well as during subsequent intracellular sorting.

Endocytosis is an essential cellular process consisting of the internalization of extracellular cargo and its transport towards different intracellular destinations. Multiple endocytic routes are tailored for the internalization and trafficking of different types of cargo and multiple endocytic organelles provide specialized biochemical environments where different molecular events take place. Membrane receptors and cargo molecules are internalized by both Clathrin-dependent and –independent endocytosis into early endosomes. From here two main endocytic routes are followed: 1) the recycling route, mainly followed by membrane receptor and other molecules like Transferrin, brings the cargo back to the plasma membrane and 2) the degradative route, followed by molecules like Epidermal Growth Factor (EGF) and Lipoprotein particles (LDL), leads the cargo to degradation into late endosomes/lysosomes. In addition to the basic function of intracellular cargo transport, the endocytic system fulfils many other cellular and developmental functions such as transmission of proliferative and survival signals and defence against pathogens. In order for cells to properly perform their various and numerous functions in organs and tissues, the activity of the endocytic system needs to be coordinated between cells and, within individual cells, integrated with other cellular functions. Even though molecules orchestrating the endocytic sorting and transport of different types of cargo have long been investigated, our understanding of the molecular machinery underlying endocytosis and its coordination into the cellular systems remains fragmentary. The work presented in this thesis aimed at understanding how this high-order regulation and integration is achieved. This requires not only a comprehensive analysis of molecular constituents of the endocytic system but also an understanding of the general design principles underlying its function. To this end, in collaboration with several members of the Zerial group and with the HT-Technology Development Studio (TDS) at MPI-CBG, I developed a new strategy to accurately profile the activity of human genes with respect to Transferrin (Tfn) and Epidermal Growth Factor (EGF) endocytosis by combining genome-wide RNAi with several siRNA/esiRNA per gene, automated high-resolution confocal microscopy, quantitative multi-parametric image analysis and high-performance computing. This provided a rich and complex genomic dataset that was subsequently subjected to analysis with a combination of tools such as a multi-parametric correlation of oligo profiles, phenotypic clustering and pathways analysis, and a Bayesian network reconstruction of key endocytic features. Altogether, the genomic endeavour and the subsequent analyses provided a number of important results: first, they revealed a much higher extent of off-target effects from RNAi and provided novel tools to infer the specific effects of genes loss of function; second, they identified a large number of novel molecules exerting a regulatory role on the endocytic system, including uncharacterized genes and genes implicated in human diseases; third, they uncovered the regulatory activity of signalling pathways such as Wnt, Integrin, TGF-β, and Notch, and found new genes regulating the sorting of cargo to a specialized subset of early endosomes that function as intracellular signalling platforms; and fourth, a systems analysis by Bayesian networks revealed that the cell specifically regulates the number, size, concentration of cargo and intracellular position of endosomes, thus uncovering novel properties of the endocytic system. In conclusion, the work presented here not only provided a dataset extremely rich of information whose potential has just begun to be uncovered but also shows how genomic datasets can be used to reveal design principles governing the functioning of biological processes.

In the post smallpox era, vaccinia virus (VACV) has emerged as an important candidate vaccine vector. As yet, the binding receptors and entry mechanisms utilised by the two infectious forms, IMV and EEV, in dendritic cells (DCs) are unknown. We have investigated the interactions between VACV and C-type lectin receptors (CLRs) that are known to be utilised by many other viruses for binding and entry in DCs. Using a variety of CLR ligands and inhibitors we were unable to inhibit IMV or EEV binding to MDDCs and we conclude that they do not bind to CLRs. We have also investigated VACV entry in MDDCs and show that both IMV and EEV enter MDDCs via an endocytic pathway. Using a variety of drugs that inhibit cellular processes we found IMV and EEV entry to be actin- and calcium-dependent. EEV entry was also cholesterol- and energy-dependent, whereas IMV entry was only partially dependent on these factors. Both IMV and EEV colocalised with endolysosomal markers. This data suggests that EEV may enter DCs via caveolin-mediated endocytosis whereas IMV entry can occur via multiple complementary mechanisms, including endocytosis and fusion. Macropinocytosis may also constitute a minor route of entry for IMV as entry was partially inhibited by dimethyl amiloride and the virus colocalised with dextran. Finally we have provided a comprehensive flow cytometric analysis of Toll-like receptor (TLR) expression at the protein level in MDDCs and monocyte-derived Langerhans cells (MDLCs) as models for different myeloid DC subsets. We found TLR expression to be cell type-specific and MDDCs expressed the full repertoire of TLRs 1-9, including small amounts of TLR8 and TLR9 on the cell surface. The expression of these TLRs that recognise nucleic acids on the surface of cells may constitute an early warning system for signalling the presence of viral invaders that would normally subvert the function of DCs. We also found TLR expression in mature cells to be dependent on the nature of the maturation stimulus (lipopolysaccharide versus cytokine/prostaglandin cocktail) and VACV infection induced profound down-regulation of all TLRs. These findings will have important implications for the rational design of VACV-vectored vaccines.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 3 uppsatser.

Thesis (Ph.D.)--Medical College of Ohio, 2005. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: William Maltese. Includes abstract. Document formatted into pages: iv, 134 p. Title from title page of PDF document. Bibliography: pages 107-132.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 121-132).

Les étapes précoces de l’infection virale sont des évènements critiques dans le déroulement du cycle viral. Notamment, l’entrée virale est la première étape d’interaction entre un virus et une cellule permettant d’initier, de maintenir et de propager l’infection. De ce fait, cette étape constitue une cible majeure de la réponse immunitaire adaptative de l’hôte. C’est ainsi que cette étude bipartite s’est intéressée aux étapes précoces de l’infection du virus de l’hépatite C (HCV). Dans un premier temps, l’obtention de clones de cellules Huh-7.5 n’exprimant plus le récepteur néonatal des immunoglobulines, le FcRn, a permis d’analyser l’implication de ce récepteur dans la neutralisation du HCV par des anticorps neutralisants. Les résultats obtenus nous informent que le récepteur FcRn n’intervient vraisemblablement pas dans la modulation de la neutralisation du HCV. Dans un second temps, nous avons réalisé une étude préliminaire afin d’approfondir les mécanismes régissant l’endocytose du HCV : à savoir, quelles sont les protéines adaptatrices responsables du déclenchement de l’endocytose dépendante de la clathrine et s’il existe une potentielle voie d’entrée alternative pour ce virus. A ces fins, nous avons opté pour une stratégie basée sur la transfection de siRNA, couplée à l’utilisation des pseudoparticules HCVpp qui constituent une approche encore pertinente appliquée à l’étude de l’entrée du HCV. / The early steps during a viral infection are critical events in the course of the viral cycle. Particularly, the viral entry is the first step allowing the interaction between a virus and a cell to initiate, maintain and propagate an infection. Therefore, this very step is a major target for the host adaptive immunity. This bipartite study is focused on the early steps of the infection by the hepatitis C virus (HCV). First of all, generation of Huh-7.5 clones whose expression of the neonatal Fc receptor, FcRn, has been deleted gave us the opportunity to analyze the involvement of this receptor during the neutralization of HCV by neutralizing antibodies. Regarding the results, it appears unlikely that the FcRn modulates the neutralization of HCV. Then we conducted a preliminary study to further explore the mechanisms underlying the endocytosis of HCV: that is, which are the adaptor proteins that trigger the clathrine-mediated endocytosis and if there is another putative entry pathway for the virus. To these ends we opted for a RNAi based strategy coupled to the use of HCV-derived pseudo-particles (HCVpp) that are still useful and relevant tools dedicated to HCV-entry studies.