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Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-ß/Smad pathway of fibrosis in human keratocytes in vitro
Tomislav Šarenac, Martin Trapečar, Lidija Gradišnik, Marjan Rupnik, Dušica Pahor, 2016, izvirni znanstveni članek

Opis: Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.
Ključne besede: cornea, wounds, treatment, antifibrotic treatment, keratocytes
Objavljeno: 23.06.2017; Ogledov: 14; Prenosov: 0
.pdf Polno besedilo (1,89 MB)

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, pregledni znanstveni članek

Opis: 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.
Ključne besede: calcium sensors, membrane potential sensors, calcium imaging, membrane potential imaging, beta cell, pancreas, denoising, patch-clamp
Objavljeno: 22.06.2017; Ogledov: 9; Prenosov: 0
.pdf Polno besedilo (4,17 MB)

Adapted user-centered design
Emilija Stojmenova, Bojan Imperl, Tomaž Žohar, Dejan Dinevski, 2012, izvirni znanstveni članek

Opis: Being familiar with all the benefits of e-Health and the strategic plan for the Slovenian health sectors informatization, Telekom Slovenia and the Faculty of Medicine from the University of Maribor, along with other partners, have initiated an e-Health project. The project group is developing various e-Health services that are based on modern ICT (information and communications technology) solutions and will be available on several screens. In order to meet the users needs and expectations and, consequently, achieve the high acceptance of e-Health services, the user-centered design (UCD) approach was employed in the e-Health project. However, during the research it was found that conventional UCD methods are not completely appropriate for older adults: the target population of the e-Health services. That is why the selected UCD methods were modified and adapted for older adults. The modified UCD methods used in the research study are presented in this paper. Using the results of the adapted UCD methods, a prototype for a service named MedReminder was developed. The prototype was evaluated by a group of 12 study participants. The study participants evaluated the MedReminder service as acceptable with a good potential for a high adoption rate among its target population, i.e., older adults.
Ključne besede: ICT, e-Health, MedReminder, user-centered design, modification, adaptation, older adults
Objavljeno: 21.06.2017; Ogledov: 9; Prenosov: 0
.pdf Polno besedilo (1,04 MB)

The SOS response master regulator LexA is associated with sporulation, motility and biofilm formation in Clostridium difficile
Beata Maria Walter, Stephen Cartman, Nigel Peter Minton, Matej Butala, Maja Rupnik, 2015, izvirni znanstveni članek

Opis: The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.
Ključne besede: toxins, bacterial sporulation, DNA damage, antibiotics
Objavljeno: 19.06.2017; Ogledov: 30; Prenosov: 0
.pdf Polno besedilo (6,06 MB)

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, izvirni znanstveni članek

Opis: 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.
Ključne besede: glucose, pancreas, mice
Objavljeno: 19.06.2017; Ogledov: 27; Prenosov: 0
.pdf Polno besedilo (2,57 MB)

The analysis of intracellular and intercellular calcium signaling in human anterior lens capsule epithelial cells with regard to different types and stages of the cataract
Marko Gosak, Rene Markovič, Aleš Fajmut, Marko Marhl, Marko Hawlina, Sofija Andjelić, 2015, izvirni znanstveni članek

Opis: In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of LECs in cataractogenesis.
Ključne besede: intracellular calcium signaling, anterior lens, epithelial cells, cataract
Objavljeno: 19.06.2017; Ogledov: 27; Prenosov: 0
.pdf Polno besedilo (1,45 MB)

Sequence similarity of Clostridium difficile strains by analysis of conserved genes and genome content is reflected by their ribotype affiliation
Hedwig Kurka, Armin Ehrenreich, Wolfgang Ludwig, Marc Monot, Maja Rupnik, Frédéric Barbut, Alexander Indra, Bruno Dupuy, Wolfgang Liebl, 2014, izvirni znanstveni članek

Opis: PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation.
Ključne besede: clostridium difficile, comparative genomics, RNA polymerase
Objavljeno: 19.06.2017; Ogledov: 23; Prenosov: 0
.pdf Polno besedilo (1,35 MB)

Recombination drives evolution of the Clostridium difficile 16S-23S rRNA intergenic spacer region
Sandra Janežič, Alexander Indra, Thomas Rattei, Thomas Weinmaier, Maja Rupnik, 2014, izvirni znanstveni članek

Opis: PCR-ribotyping, a typing method based on size variation in 16S-23S rRNA intergenic spacer region (ISR), has been used widely for molecular epidemiological investigations of C. difficile infections. In the present study, we describe the sequence diversity of ISRs from 43 C. difficile strains, representing different PCR-ribotypes and suggest homologous recombination as a possible mechanism driving the evolution of 16S-23S rRNA ISRs. ISRs of 45 different lengths (ranging from 185 bp to 564 bp) were found among 458 ISRs. All ISRs could be described with one of the 22 different structural groups defined by the presence or absence of different sequence modules; tRNAAla genes and different combinations of spacers of different lengths (33 bp, 53 bp or 20 bp) and 9 bp direct repeats separating the spacers. The ISR structural group, in most cases, coincided with the sequence length. ISRs that were of the same lengths had also very similar nucleotide sequence, suggesting that ISRs were not suitable for discriminating between different strains based only on the ISR sequence. Despite large variations in the length, the alignment of ISR sequences, based on the primary sequence and secondary structure information, revealed many conserved regions which were mainly involved in maturation of pre-rRNA. Phylogenetic analysis of the ISR alignment yielded strong evidence for intra- and inter-homologous recombination which could be one of the mechanisms driving the evolution of C. difficile 16S-23S ISRs. The modular structure of the ISR, the high sequence similarities of ISRs of the same sizes and the presence of homologous recombination also suggest that different copies of C. difficile 16S-23S rRNA ISR are evolving in concert
Ključne besede: clostridium difficile, sequence analysis, ribosomal RNS
Objavljeno: 19.06.2017; Ogledov: 8; Prenosov: 0
.pdf Polno besedilo (846,32 KB)

Rab3a is critical for trapping alpha-MSH granules in the high Ca2+-affinity pool by preventing constitutive exocytosis
Simon Sedej, Maša Skelin, Oliver Schlüter, Marjan Rupnik, 2013, izvirni znanstveni članek

Opis: Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, -melanocyte stimulating hormone (-MSH) and -endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that -MSH, but not -endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of -MSH. Increased constitutive secretion of -MSH from incubated tissue slices was associated with reduced -MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of -MSH.
Ključne besede: melanotrophs, exocytosis
Objavljeno: 19.06.2017; Ogledov: 8; Prenosov: 0
.pdf Polno besedilo (1,90 MB)

Platelet-rich plasma, especially when combined with a TGF-ß inhibitor promotes proliferation, viability and myogenic differentiation of myoblasts in vitro
Robi Kelc, Martin Trapečar, Lidija Gradišnik, Marjan Rupnik, Matjaž Vogrin, 2015, izvirni znanstveni članek

Opis: Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-β inhibitor.
Ključne besede: muscles, skeletal, injuries, TGF-beta, plasma, thrombocytes, myoblasts, fibrosis, prevention, regeneration
Objavljeno: 19.06.2017; Ogledov: 10; Prenosov: 0
.pdf Polno besedilo (2,07 MB)

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