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1.
Vpliv presnovno pomembnih dejavnikov na funkcijo celic beta
Andraž Stožer, 2013, dissertation

Abstract: V endokrinih celicah Langerhansovih otočkov so kalcijevi ioni (Ca2+) pomemben element sklopitve med celično stimulacijo in sekrecijo hormonov. Od uvedbe sodobne fluorimetrije so številne študije in vitro, pretežno na izoliranih otočkih, preučevale učinke različnih sekretagogov na znotrajcelično koncentracijo ionov Ca2+, predvsem v celicah beta, ki izločajo inzulin. Pri razumevanju fizioloških sprememb znotrajcelične koncentracije ionov Ca2+ ostaja vrsta nejasnosti. Zaradi tehničnih omejitev izsledki teh študij temeljijo na podatkih, pridobljenih na razmeroma majhnem številu v najbolj zunanjih plasteh otočka nahajajočih se celic. Prav tako so rezultati odvisni od trajanja in pogojev kultivacije izoliranih otočkov, zaradi česar niso zmeraj združljivi z rezultati, pridobljenimi v pogojih in vivo. Poglavitni nejasnosti sta, kakšna je narava oscilacij znotrajcelične koncentracije ionov Ca2+ in ali so kalcijevi valovi tisti, ki sinhronizirajo aktivnost velikega števila celic. V tej disertaciji predstavljamo novo eksperimentalno metodo in situ, ki združuje pripravo sveže tkivne rezine trebušne slinavke miši z barvanjem z estrskimi fluorescenčni indikatorji, občutljivimi na znotrajcelično koncentracijo ionov Ca2+, in s konfokalno mikroskopijo z laserskim skenerjem. Metodo smo razvili, da bi v eksperimentalnih pogojih, ki so zelo blizu razmeram in vivo, preučevali veliko število celic iz vseh plasti Langerhansovih otočkov in z njeno pomočjo odgovorili na zgornji vprašanji. Da bi dobili boljši vpogled v aktivnost in sklopitev celic, smo v pričujoči nalogi kot prvi analizirali delovanje Langerhansovih otočkov s pomočjo teorije kompleksnih mrež. V realni prostor umeščene mreže smo konstruirali iz električno sklopljenih celic beta, ki smo jih funkcionalno povezali na osnovi korelacij med fluorescenčnimi signali vseh parov celic. Funkcionalne mreže smo analizirali v luči znanih strukturnih in fizioloških značilnosti otočkov. Naši poskusi so pokazali, da so v svežih tkivnih rezinah stabilne hitre oscilacije koncentracije znotrajceličnih ionov Ca2+ prevadujoči tip oscilacij in da so kalcijevi valovi podlaga sinhronizacije teh oscilacij v različnih celicah določenega otočka. Prav tako smo ugotovili, da na začetku odziva na glukozo tudi velika sprememba znotrajcelične koncentracije Ca2+ v določeni celici ni zadosten pogoj za odziv ostalih celic in da je za sklopitev med celicami nujna presnovna aktivacija vsake celice. Ob preučevanju razvoja mreže celic beta po odzivu na glukozo smo pokazali, da so celice bolj korelirane v stimulativnih kot v nestimulativnih pogojih. Največjo stopnjo korelacije, v pretežni meri neodvisno od evklidskih razdalj med celicami, smo ugotovili v času aktivacije in deaktivacije, ko se vse celice odzivajo na zunanji stimulus. V času stacionarne aktivnosti stopnja korelacije kaže jasno odvisnost od evklidske razdalje in potrjuje idejo, da se celice beta sinhronizirajo s pomočjo kalcijevih valov, ki se širijo od celice do celice. Funkcionalna mreža celic beta kaže lastnosti malega sveta, kar namiguje, da je sincicij celic beta organiziran tako, da podpira optimalno sinhronizacijo. Naša metoda je omogočila vpogled v delovanje večjega števila celic naenkrat kot doslej razpoložljive metode, odpravila kontroverze glede tipa oscilacij in prisotnosti kalcijevih valov in osvetlila razmerje med topologijo mreže in njenim delovanjem. Menimo, da so to zadostni razlogi, da postane ena od metod izbora za študij fiziologije Langerhansovih otočkov.
Keywords: celica beta, sinhronizacija, teorija grafov, kompleksna mreža, znotrajcelični kalcij, oscilacije
Published: 12.03.2013; Views: 1636; Downloads: 360
.pdf Full text (8,17 MB)

2.
IZDELAVA ELEKTROKARDIOGRAFA IN DIAGNOSTIKA POTENCIALNIH OBOLENJ SRCA
Gregor Nikolić, 2014, undergraduate thesis

Abstract: V diplomski nalogi je opisan razvoj in uporaba tri-točkovnega elektrokardiografa, s katerim merimo razlike potencialov napetosti na telesu, ki jih povzroča srce v svoji okolici. Predstavljeno je osnovno delovanje srca ter predstavitev srca kot električni dipol, s katerim opisujemo povzročene potenciale. Elektrokardiograf je izdelan iz analognega in digitalnega dela vezja ter programskega vmesnika na računalniku, s katerim opazujemo zajet signal ter izvajamo diagnostiko potencialnih obolenj srca. V analognem delu se soočamo z zajemanjem izmeničnega signala, ki je izredno majhne amplitude, ki je superponiran na razmeroma visok enosmerni signal. V digitalnem delu pa se soočamo s pretvorbo analognega signala in komunikacijo z računalnikom. K digitalnemu delu lahko pripišemo tudi razvoj programskega vmesnika, ki omogoča interpretacijo signala na zaslon in diagnostiko. Tako za analogni kot digitalni del smo opisali razvoj, delovanje in uporabo.
Keywords: elektrokardiograf, EKG, diagnostika, obolenja, srce
Published: 19.02.2015; Views: 821; Downloads: 202 
(1 vote)
.pdf Full text (3,18 MB)

3.
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)

4.
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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5.
Akutna respiracijska acidoza in alkaloza
Marjan Rupnik, Andraž Stožer, 2014, review article

Abstract: Izhodišče: Obstajajo trije različni pristopi k oceni kislinsko-baznega stanja bolnika: bostonski, kopenhagenski in Stewartov, ki temeljijo na različnih izmerjenih parametrih. Poleg vnetih debat o tem, kateri od pristopov je boljši, vnaša med študente, raziskovalce in klinike zmedo dejstvo, da med svojim študijem tipično osvojijo le enega od omenjenih pristopov in zato ne morejo v celoti razumeti virov, ki uporabljajo katerega od drugih pristopov, poleg tega pa ne morejo kritično oceniti prednosti in omejitev določenega pristopa. Avtorja v članku predstaviva in opredeliva osnovne parametre, značilne za posamezne pristope, in poudariva razlike in podobnosti med njimi. Posebno pozornost namenjava vprašanju, kako posamezni pristopi ocenjujejo spremembo v koncentraciji plazemskega bikarbonata, do katere pride med primarnimi respiracijskimi spremembami in katerih ustrezno razumevanje je potrebno za pravilno interpretiranje kroničnih respiracijskih in metaboličnih kislinsko-baznih sprememb. Zaključek: Med akutno respiracijsko acidozo se koncentracija bikarbonata zviša in med akutno respiracijsko alkalozo zniža v odvisnosti od moči nebikarbonatnih pufrov. Med akutnimi respiracijskimi motnjami se koncentracija pufrske baze (uporablja jo kopenhagenski pristop), navidezna razlika močnih ionov, efektivna razlika močnih ionov in vrzel močnih ionov (uporablja jih Stewartov pristop) ne spremenijo, anionska vrzel (uporabljata jo bostonski in kopenhagenski pristop) pa se med akutno respiracijsko acidozo zmanjša, med akutno respiracijsko alkalozo pa zveča.
Keywords: respiratorne motnje, Davenportov diagram, Gamblov diagram, kislinsko-bazna motnja
Published: 30.12.2015; Views: 481; Downloads: 24
.pdf Full text (780,41 KB)

6.
Fiziologija prebavne cevi
Andraž Stožer, Marjan Rupnik, Jurij Dolenšek, 2010, review article

Keywords: fiziologija, prebava, prebavila, prebavna cev
Published: 30.12.2015; Views: 317; Downloads: 16
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7.
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: 371; Downloads: 21
URL Full text (0,00 KB)

8.
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: 175; Downloads: 9
.pdf Full text (957,14 KB)

9.
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: 151; Downloads: 15
.pdf Full text (4,17 MB)

10.
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: 243; Downloads: 10
.pdf Full text (798,57 KB)

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