20.500.12556/RUL-124106 Elementarni dogodki eksocitoze in endocitoze v aktiviranih astrocitih v kulturi doktorska disertacija Elementary exocytotic and endocytotic events in activated cultured astrocytes doctoral dissertation Astrociti so homeostatične celice centralnega živčevja, ki se odzivajo na patološke dražljaje s procesom aktivacije. Vnetni citokin interferon γ (IFNγ) v astrocitih inducira izražanje molekul poglavitnega histokompatibilnostnega kompleksa razreda II (MHCII), ki so potrebne za predstavitev antigenov. Izolirane astrocite tradicionalno gojimo v celični kulturi s serumom, ki poveča bazalno raven aktivacije in spremeni njihovo morfologijo ter znotrajcelično signalizacijo s kalcijevimi ioni. Vpliv obeh prožilcev aktivacije astrocitov na elementarne dogodke ekso-/endocitoze v astrocitih je nepoznan. V delu smo zato raziskali dinamiko in lastnosti elementarnih dogodkov ekso-/endocitoze v podganjih netretiranih serumskih astrocitih, serumskih astrocitih, tretiranih z IFNγ in astrocitih v brezserumski kulturi. Znotrajcelično porazdelitev imunofluorescenčno označenih molekul MHCII smo analizirali s konfokalno mikroskopijo in mikroskopijo s strukturirano osvetlitvijo, lastnosti elementarnih dogodkov ekso-/endocitoze smo raziskali z meritvami membranske kapacitivnosti z metodo vpete napetosti krpice membrane. V astrocitih, tretiranih z IFNγ, so se molekule MHCII lokalizirale pretežno v pozne endo-/lizosome, vendar so dosegle tudi plazmalemo. Z njo so prehodno interagirali večji eksocitotski mešički, medtem ko so bili inhibirani dogodki popolne endocitoze. Ekso-/endocitotska dejavnost brezserumskih astrocitov je bila primerljiva z dejavnostjo serumskih astrocitov v spontanih razmerah, medtem ko je bil zavrt njihov ekso-/endocitotski odziv na draženje z ATP. Aktivacija astrocitov v kulturi z IFNγ ali serumom spremeni dinamiko elementarnih dogodkov ekso-/endocitoze in podaljša zadrževanje molekul MHCII na površini. Astrocytes are homeostatic glial cells in the central nervous system that respond to pathologic changes with a process of activation. Interferon γ (IFNγ), an inflammatory cytokine, induces astrocyte expression of major histocompatibility class II (MHCII) molecules required for antigen presentation. Isolated astrocytes are traditionally cultured with serum; however, serum increases astrocyte basal activation and alters their morphology and intracellular calcium signalling. The impact of both triggers of astrocyte activation on exo-/endocytotic activity in astrocytes is not understood. We have thus studied the properties and dynamics of exo-/endocytotic events in non-treated and IFNγ-treated serum as well as serum-free cultured rat astrocytes. The intracellular distribution of immunofluorescently labelled MHCII molecules was analysed with confocal and structural illumination microscopies. Furthermore, elementary exo-/endocytotic activity was studied by patch clamp membrane capacitance measurements. In IFNγ-treated astrocytes, MHCII molecules predominantly localized to late endo-/lysosomes, but also reached the plasmalemma. Additionally, larger exocytotic vesicles reversibly interacted with the plasmalemma, while full endocytotic events were inhibited. Exo-/endocytotic activity of astrocytes in serum-free culture was unaltered at rest, however their exo-/endocytotic activity in response to ATP stimulation was suppressed. Activated cultured astrocytes instigated by treatment with IFNγ or serum culture supplementation display altered dynamics of elementary exo-/endocytotic activity and prolonged surface retention of MHCII. astrociti eksocitoza endocitoza aktivacija astrocitov IFNγ MHCII predstavitev antigenov serum elektrofiziologija meritve kapacitivnosti astrocytes exocytosis endocytosis astrocyte activation IFNγ MHCII antigen presentation serum electrophysiology capacitance measurements true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2020-12-31 07:15:21 2020-12-31 07:15:28 2022-09-04 03:57:43 0000-00-00 00:00:00 2020 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 187597 49776643 11639.pdf 11639.pdf 1 3152DDD9FE05F280976946A9933F1735 9269685d6480d15359e47be7b72109cfc17ab9696bafea5c11ae4e554afbbadc 7a7d2f45-a1bb-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=139494 Biotehniška fakulteta 0 0 0