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Okužbe drenažnih sistemov v nevrokirurgiji
Jasmina Golc, Jasna Zakelšek, 2011, research project (high school)

Published: 04.01.2012; Views: 1447; Downloads: 55
.pdf Full text (503,67 KB)

Analiza genetskih dejavnikov pri bolnikih povezanih s pristnostjo HLA-B27
Jasmina Rehar, 2011, research project (high school)

Published: 04.01.2012; Views: 1599; Downloads: 163
.pdf Full text (643,74 KB)

Spatial network representation of complex living tissues
Dean Korošak, Marjan Rupnik, 2008, published scientific conference contribution

Abstract: Networks were widely used to describe organizational and functional principles of living organisms across various scales. The topology of such biological complex networks often turned out to be "scale-free", with the power-law distribution of number of links per node, robust and modular with underlying self-similar structure. However, the topology of cytoarchitecture in living tissues has not yet received wide attention from the network perspective. Here we discuss the spatial complex network model of coupled clusters of beta cells in pancreatic islets. Networks of cells in pancreatic islets were constructed from the 2D section images presenting fluorescently labelled intercellular spaces obtained by two-photon laser scanning microscopy of whole pancreas tissue slices, and cells conductances measured electrophysiologically using whole-cell patch-clamp. We find that the heterogeneity of beta cells in intact living islets induces scale-free topology of the tissue network. Furthermore, we show that the islet-like structures visually similar to 2D section images can be obtained using Voronoi diagrams of random points.
Keywords: pancreatic islets, betta cells, complex networks, cytoarchitecture
Published: 31.05.2012; Views: 962; Downloads: 3
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Calcium dependencies of regulated exocytosis in different endocrine cells
Jurij Dolenšek, Maša Skelin, Marjan Rupnik, 2011, review article

Abstract: Exocytotic machinery in neuronal and endocrine tissues is sensitive to changesin intracellular Ca2+ concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca2+ sensitivity in endocrine cells, accurate and fast determination of Ca2+ dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca2+ concentration ([Ca2+]i) that leads to a robust exocytotic activity. Slow increases in the [Ca2+]i revealed exocytotic phases with different Ca2+ sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca2+ sensitivity ćclassesć of secretory vesicles have been observed: a highly Ca2+-sensitive, amedium Ca2+-sensitive and a low Ca2+- sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca2+ sensitivity classes. This significantly alterslate steps in the secretory release of hormones even without utilizationof an additional Ca2+ sensor protein.
Keywords: Calcium sensitivity, Exocytosis, Insulin-secreting cells, Chromaffin cells, Melanotrophs
Published: 05.06.2012; Views: 737; Downloads: 10
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Exocytosis of insulin in vivo maturation of mouse endocrine pancreas
Aldo Rozzo, Tiziana Meneghel-Rozzo, Saška Lipovšek Delakorda, Shi-Bing Yang, Marjan Rupnik, 2009, published scientific conference contribution

Abstract: The aim of this study was to define when an insulin-positive cell becomes functional in vivo and starts to exocytose insulin in a regulated nutrient-dependent manner. Insulin-positive cells appear in embryonic life (midgestation) and complete their maturation, presumably around birth. In order to work with embryonic and newborn endocrine pancreas, we used organotypic slices. The mouse embryonic pancreas slices show high basal insulin release that is not further elevated by high glucose levels. Despite the presence of functional voltage-activated ion channels, the cells are not electrically active in the presence of secretagogues. At birth, the high basalinsulin release drops and, after postnatal day 2, the insulin-positive cells show both adult-like bursting electrical activity and hormone release induced by high glucose levels. These properties allowed us to define them as beta cells. Despite the apparent stability of the transcription factor profile reported in insulin-positive cells during late-embryonic life, functional beta cells appear only 2 days after birth.
Keywords: cell biology, insulin release, embrios, newborn, beta-cell maturation, developing pancreas
Published: 07.06.2012; Views: 528; Downloads: 5
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Maša Skelin, 2011, dissertation

Abstract: Raziskovali smo vlogo proteinskih kinaz v procesu uravnavane eksocitoze v celicah beta trebušne slinavke. Za stimulacijo od Ca2+ odvisne sekrecije smo uporabili vlak depolarizacijskih pulzov ali počasno fotolizo, s katerima smo kontrolirali znotrajcelično aktivnost Ca2+ ionov. S pomočjo meritev kapacitivnosti celične membrane smo zasledovali eksocitozo, to je zlivanje sekretornih mešičkov s plazemsko membrano. Pri kontroli je zadostna sprememba Ca2+ sprožila vsaj dve fazi eksocitoze. Hitrost spremembe kapacitivnosti je v odvisnosti od [Ca2+]i razkrila saturacijsko kinetiko z visoko kooperativnostjo, pri čemer je bila polovica maksimalne hitrosti dosežena pri 2,9 ± 0,2 µM kalcija. Nato smo z dodajanjem cAMP ugotavljali vlogo PKA v procesu eksocitoze. Naši rezultati kažejo, da cAMP stimulira eksocitozo pri značilno nižji [Ca2+]i v primerjavi s kontrolo. Enake rezultate smo dobili, ko smo s 6-Phe-cAMP direktno stimulirali PKA, medtem ko aktivacija Epac2, ki je prav tako ena izmed poti delovanja cAMP, ni imela posebnega vpliva na spremembo občutljivosti sekretornega aparata. V nadaljevanju smo preučili še vlogo PKC in Cdk5 v procesu izločanja inzulina. Aktivacija PKC je znižala občutljivost prve faze eksocitoze v primerjavi z inhibicijo PKC, medtem ko je inhibicija Cdk5 zmanjšala hitrost zlivanja sekretornih mešičkov s plazemsko membrano, pri čemer je ostala občutljivost na [Ca2+]i nespremenjena. Ker literatura navaja, da PKC in Cdk5 fosforilirata vrsto proteinov, vključenih v mehanizem sekrecije, smo testirali vlogo Munc18-1, ki je po našem mnenju ena najbolj verjetnih fosforilacijskih tarč PKC in Cdk5. Povišana ekspresija PKC fosforilacijske mutante Munc18-1 je značilno znižala amplitudo prve faze eksocitoze in zvišala njeno občutljivost na [Ca2+]i. Amp1 je bila značilno nižja tudi pri povišani ekspresiji Cdk5 fosforilacijske mutante Munc18-1, vendar je bila njena občutljivost na [Ca2+]i tokrat značilno nižja. Prav tako je bila značilno nižja tudi hitrost zlivanja mešičkov s plazemsko membrano. Naši rezultati tako potrjujejo hipotezo, da cAMP v celicah beta spremeni občutljivost sekretornega aparata na Ca2+ ione z aktivacijo PKA. Tudi PKC in Cdk5 sta z delovanjem preko proteina Munc18-1 pomembna regulatorja eksocitoze, pri čemer je PKC pomembna za preprečevanje zlivanja mešičkov s plazemsko membrano pri nezadostni [Ca2+]i, Cdk5 pa sodeluje v pripravi mešičkov na zlitje.
Keywords: beta celice, eksocitoza, proteinske kinaze, občutljivost na kalcij, izločanje inzulina
Published: 19.03.2013; Views: 1328; Downloads: 108
.pdf Full text (13,49 MB)

Glucose-stimulated calcium dynamics in Islets of Langerhans in acute mouse pancreas tissue slices
Andraž Stožer, Jurij Dolenšek, Marjan Rupnik, 2013, original scientific article

Abstract: In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous in vitro studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings in vivo. The main controversies regard the types ofcalcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the in situ acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidencethat even a large amplitude calcium signal was not sufficient and thatmetabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future.
Keywords: glucose, pancreas, islets of Langerhans, mice
Published: 10.07.2015; Views: 290; Downloads: 10
.pdf Full text (2,83 MB)

ATP regulates sodium channel kinetics in pancreatic islet beta cells
Na Zou, Marjan Rupnik, 2013, original scientific article

Abstract: Pancreatic beta cells act as glucose sensors, in which intracellular ATP ([ATP](i)) are altered with glucose concentration change. The characterization of voltage-gated sodium channels under different [ATP](i) remains unclear. Here, we demonstrated that increasing [ATP](i) within a certain range of concentrations (2-8 mM) significantly enhanced the voltage-gated sodium channel currents, compared with 2 mM cytosolic ATP. This enhancement was attenuated by even high intracellular ATP (12 mM). Furthermore, elevated ATP modulated the sodium channel kinetics in a dose-dependent manner. Increased [ATP](i) shifted both the current-voltage curve and the voltage-dependent inactivation curve of sodium channel to the right. Finally, the sodium channel recovery from inactivation was significantly faster when the intracellular ATP level was increased, especially in 8 mM [ATP](i), which is an attainable concentration by the high glucose stimulation. In summary, our data suggested that elevated cytosolic ATP enhanced the activity of Na(+) channels, which may play essential roles in modulating cell excitability and insulin release when blood glucose concentration increases.
Keywords: Langerhansovi otočki, trebušna slinavka
Published: 10.07.2015; Views: 476; Downloads: 7
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Is tissue augmentation a reality in biosurgery? An experimental study of endothelial cell invasion into tissue filler
Tomaž Velnar, Vladimir Smrkolj, Marjan Rupnik, Lidija Gradišnik, 2013, original scientific article

Abstract: New therapeutic approaches for wound treatment are evolving. Non healing wounds in oncology and after trauma may be cured by a novel technique of tissue augmentation with soft tissue fillers. The principle resides in filling the wound with collagen filler in order to seal the defect and promote healing. Successful angiogenesis forms the basis of tissue filler survival and determines the outcome of the healing process. During this study, basic data about endothelial cell invasion into collagen-made substratum was collected that could be used for neoangiogenesis studies in tissue augmentation techniques for large wound defect treatment. In the in vitro assay, the human umbilical vein endothelial cells (HUVEC) grow into a three-dimensional framework of collagenous tissue fillers, forming the basic step for angiogenesis. After heparins were used as chemotactic agents, a typical bell-shaped relationship between chemotaxis and agent concentrations was found. Significant cell infiltration was present in the assays with chemotactic agents. These observations support the potential for tissue augmentation with soft tissue fillers that could be used in acute and chronic non healing traumatic and oncology wounds after extensive surgical resections and radiotherapy.
Keywords: angiogenesis, cell invasion, tissue augmentation, tissue filler, wound healing
Published: 10.07.2015; Views: 404; Downloads: 6
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Cell physiology in tissue slices
Maša Skelin, Andraž Stožer, Jurij Dolenšek, Marjan Rupnik, 2013, review article

Abstract: Uporaba izoliranih endokrinih celic je omogočila razvoj številnih metod za oceno osnovne celične arhitekture in opredelitev pomembnega dela molekularnih mehanizmov, ki sodelujejo pri celičnih procesih, kot sta vzdražnost celic in izločanje hormonov v procesu eksocitoze po porastu znotrajcelične koncentracije kalcijevih ionov. Ta prizadevanja so vodila v oblikovanje dogovornih modelov, ki razlagajo aktivacijo in splošen način delovanja določenega celičnega tipa. Z uporabo svežih tkivnih rezin lahko presežemo dosedanje raziskave in razkrijemo celo vrsto porajajočih se lastnosti, ki jih dogovorni modeli ne morejo zlahka predvideti niti razložiti. V tem članku predstavljamo izbor naših najpomembnejših eksperimentalnih ugotovitev na celicah beta v tkivnih rezinah trebušne slinavke.
Keywords: celica beta, Langerhansov otoček, tkivna rezina trebušne slinavke miši, elektrofiziologija, konfokalna fluorescenčna mikroskopija, kalcijevo barvilo, teorija grafov
Published: 10.07.2015; Views: 367; Downloads: 20
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