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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: 741; Downloads: 12
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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: 294; Downloads: 10
.pdf Full text (2,83 MB)

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: 370; Downloads: 21
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Fiziologija prebavne cevi
Andraž Stožer, Marjan Rupnik, Jurij Dolenšek, 2010, review article

Keywords: fiziologija, prebava, prebavila, prebavna cev
Published: 30.12.2015; Views: 318; Downloads: 16
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Fiziologija prebavne cevi
Andraž Stožer, Marjan Rupnik, Jurij Dolenšek, 2010, review article

Keywords: Bolezni prebavil, fiziologija, prebava, prebavila, prebavna cev, Prebavne cevi, Prehrana
Published: 30.12.2015; Views: 375; Downloads: 21
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Progressive glucose stimulation of islet beta cells reveals a transition from segregated to integrated modular functional connectivity patterns
Marjan Rupnik, Andraž Stožer, Marko Gosak, Rene Markovič, Marko Marhl, Jurij Dolenšek, 2015, original scientific article

Abstract: Collective beta cell activity in islets of Langerhans is critical for the supply of insulin within an organism. Even though individual beta cells are intrinsically heterogeneous, the presence of intercellular coupling mechanisms ensures coordinated activity and a well-regulated exocytosis of insulin. In order to get a detailed insight into the functional organization of the syncytium, we applied advanced analytical tools from the realm of complex network theory to uncover the functional connectivity pattern among cells composing the intact islet. The procedure is based on the determination of correlations between long temporal traces obtained from confocal functional multicellular calcium imaging of beta cells stimulated in a stepwise manner with a range of physiological glucose concentrations. Our results revealed that the extracted connectivity networks are sparse for low glucose concentrations, whereas for higher stimulatory levels they become more densely connected. Most importantly, for all ranges of glucose concentration beta cells within the islets form locally clustered functional sub-compartments, thereby indicating that their collective activity profiles exhibit a modular nature. Moreover, we show that the observed non-linear functional relationship between different network metrics and glucose concentration represents a well-balanced setup that parallels physiological insulin release.
Keywords: endocrinology, computational biophysics, calcium signalling, biological physics
Published: 23.06.2017; Views: 178; Downloads: 10
.pdf Full text (957,14 KB)

Membrane potential and calcium dynamics in beta cells from mouse pancreas tissue slices
Marjan Rupnik, Andraž Stožer, Jurij Dolenšek, Borut Žalik, Maša Skelin, Marko Gosak, Denis Špelič, 2015, review article

Abstract: Beta cells in the pancreatic islets of Langerhans are precise biological sensors for glucose and play a central role in balancing the organism between catabolic and anabolic needs. A hallmark of the beta cell response to glucose are oscillatory changes of membrane potential that are tightly coupled with oscillatory changes in intracellular calcium concentration which, in turn, elicit oscillations of insulin secretion. Both membrane potential and calcium changes spread from one beta cell to the other in a wave-like manner. In order to assess the properties of the abovementioned responses to physiological and pathological stimuli, the main challenge remains how to effectively measure membrane potential and calcium changes at the same time with high spatial and temporal resolution, and also in as many cells as possible. To date, the most wide-spread approach has employed the electrophysiological patch-clamp method to monitor membrane potential changes. Inherently, this technique has many advantages, such as a direct contact with the cell and a high temporal resolution. However, it allows one to assess information from a single cell only. In some instances, this technique has been used in conjunction with CCD camera-based imaging, offering the opportunity to simultaneously monitor membrane potential and calcium changes, but not in the same cells and not with a reliable cellular or subcellular spatial resolution. Recently, a novel family of highly-sensitive membrane potential reporter dyes in combination with high temporal and spatial confocal calcium imaging allows for simultaneously detecting membrane potential and calcium changes in many cells at a time. Since the signals yielded from both types of reporter dyes are inherently noisy, we have developed complex methods of data denoising that permit for visualization and pixel-wise analysis of signals. Combining the experimental approach of high-resolution imaging with the advanced analysis of noisy data enables novel physiological insights and reassessment of current concepts in unprecedented detail.
Keywords: calcium sensors, membrane potential sensors, calcium imaging, membrane potential imaging, beta cell, pancreas, denoising, patch-clamp
Published: 22.06.2017; Views: 152; Downloads: 16
.pdf Full text (4,17 MB)

Functional connectivity in islets of Langerhans from mouse pancreas tissue slices
Jurij Dolenšek, Matjaž Perc, Marko Marhl, Marjan Rupnik, Dean Korošak, Andraž Stožer, Marko Gosak, 2013, original scientific article

Abstract: We propose a network representation of electrically coupled beta cells in islets of Langerhans. Beta cells are functionally connected on the basis of correlations between calcium dynamics of individual cells, obtained by means of confocal laser-scanning calcium imaging in islets from acute mouse pancreastissue slices. Obtained functional networks are analyzed in the light of known structural and physiological properties of islets. Focusing on the temporal evolution of the network under stimulation with glucose, we show thatthe dynamics are more correlated under stimulation than under non-stimulated conditions and that the highest overall correlation, largely independent of Euclidean distances between cells, is observed in the activation and deactivation phases when cells are driven by the external stimulus. Moreover, we find that the range of interactions in networks during activity shows a clear dependence on the Euclidean distance, lending support to previous observations that beta cells are synchronized via calcium waves spreading throughout islets. Most interestingly, the functional connectivity patterns between beta cells exhibit small-world properties, suggesting that beta cells do not form a homogeneous geometric network but are connected in a functionally more efficient way. Presented results provide support for the existing knowledge of beta cell physiology from a network perspective and shedimportant new light on the functional organization of beta cell syncitia whose structural topology is probably not as trivial as believed so far.
Keywords: islets of Langerhans, mouse pancreas
Published: 16.06.2017; Views: 244; Downloads: 11
.pdf Full text (798,57 KB)

The relationship between membrane potential and calcium dynamics in glucose-stimulated beta cell syncytium in acute mouse pancreas tissue slices
Jurij Dolenšek, Andraž Stožer, Maša Skelin, Evan Miller, Marjan Rupnik, 2013, original scientific article

Abstract: Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.
Keywords: glucose, pancreas, mice
Published: 19.06.2017; Views: 172; Downloads: 5
.pdf Full text (2,57 MB)

Dinamika cerebralne oksigenacije pri hiperventilaciji in hipoventilaciji
Eva Rotman, 2017, undergraduate thesis

Abstract: Uvod: Možgani za svoje delovanje potrebujejo stalen dotok krvi. V določenih kliničnih situacijah pa je potrebno zmanjšati pretok krvi, ob tem je nujno spremljanje količin kisika. Z raziskavo smo želeli ugotoviti, ali se lahko s tehniko bližnje infrardeče spektroskopije zazna spremembe kisika v možganih pri kratkotrajni hiperventilaciji in hipoventilaciji. Metode: V teoretičnem delu smo pridobili podatke z deskriptivno metodo dela, v empiričnem pa smo uporabili kvantitativno metodo z namenskim vzorcem. Regionalno nasičenost kisika v možganih smo izmerili s spektroskopijo v bližnjem infrardečem polju. Med preizkusom hiperventilacije in hipoventilacije smo spremljali vrednosti dihanja, pulza, PETCO2 in SpO2. Rezultati: V času hiperventilacije so se vrednosti rSO2 znižale za 2 % v primerjavi z začetnimi vrednostmi. Padanje se je nadaljevalo v času počitka 1, kljub temu, da se je PETCO2 zviševal. V času hipoventilacije počitka 3 smo opazili ponovno zviševanje rSO2 parametra. Diskusija in zaključek: S testom hiperventilacije in hipoventilacije smo prikazali vpliv PETCO2 na spreminjanje rSO2. Glavni mehanizem, ki je odgovoren za zmanjšanje rSO2 pri hiperventilaciji, je hipokapnija.
Keywords: Možganska prekrvavitev, avtoregulacija, hipokapnija, vazokonstrikcija, spektroskopija v bližnjem infrardečem polju.
Published: 13.09.2017; Views: 160; Downloads: 41
.pdf Full text (1,40 MB)

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