https://wiki.fkkt.uni-lj.si/api.php?action=feedcontributions&feedformat=atom&user=Paulahorvat 2026-04-15T21:45:23Z User contributions MediaWiki 1.39.3 https://wiki.fkkt.uni-lj.si/index.php?title=Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena,_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih&diff=18564 2021-04-20T07:48:03Z

<p>Paulahorvat: </p> <hr /> <div>== INTRODUCTION ==<br /> <br /> Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transcription factors and many other regulatory proteins are regulated in a pulsatile fashion, leading to a variety of phenotypic consequences, ranging from stress response to differentiation and signaling.[2]<br /> Pulsing has been observed in many types of proteins. In alternative bacterial sigma factors, but also in mammalian tumor suppressors like p53. Mammalian cells exhibit many pulsatile factors. The stress response pathways mediated by p53, which controls the DNA damage response pulses, just as nuclear factor κB which is involved in immune responses. [1] Iti s still unknown what functional capabilities can this pulsatile dynamic regulation provide for cells. Chemical inducers were used to dynamically regulate transgene expression. Slow degradation and metabolisation of chemical inducers has been shown as not so good application for controlling the dynamics of regulators in most pulsing systems. Therefore, more precise light-inducible systems have been developed that allow control of gene expression. Pulsatile dynamic regulation of regulators is achieved. The limitation that occurs is supplying adequate light intensity to activate these optogenetic systems. To solve this problem luminiscent opsins were developed ( directly coupling bioluminescent proteins to conventional light-sensitive opsins). BRET or bioluminescence resonance energy transfer is how luciferase substrates create bioluminiscence and activate opsins. After some time inactivation of opsins occurs and they regulate the cell activity in a pulsatile fashion. That is where the idea for transgene expression system that allows pulsatile regulation of gene expression both in vitro and in vivo came from. [2]<br /> Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. [6]<br /> <br /> == EXPERIMENT ==<br /> <br /> In this experiment the development of the luminGAVPO, a luminsecent transcription factor is explained. LuminGAVPO enables pulsatile and quantitative activation of transgene expression in both mammalian cells and animals in a furimazine- or light irradiance-dependent manner. By controlling blood glucose homeostasis in type 1 diabetic mice its usefulness is tested. [2]<br /> <br /> == RESULTS ==<br /> <br /> Design and construction of the LuminON transgene expression system.<br /> NanoLuc (Nluc) is a engineered luciferase from a deep sea shrimp, which is stable, bright, small and with sustained luminescence. It is used as luminiscence donor ( produces blue light) to activate LOV-based (The light-oxygen-voltage proteins) optogenetic modules. Nluc was fused to the N terminus of the mCherry-LOV2 fusion protein. When a protein was coexpressed with a mitochondria-anchored mVenus-Zdk protein, both mCherry and mVenus fluorescence accumulated at mitochondria under dark conditions. But the mCherry fluorescence readily diffused to the cytosol within 5 seconds after the addition of furimazine or exposure to external blue ligh. Blue light-inducible transgene expression systems based on Vivid were developed in which transcription factors bind to the promoter region ( to activate or repress gene transcription upon blue light exposure). The next step was to create a luminiscent transcription factor of a chimeric protein that consists of Nluc and a light-switchable transcription factor, GAVPO. Nluc-GAVPO fusions activated Gluc expression upon incubation with furimazine (FZ) or illumination with external blue light. On the other hand expression of Nluc and GAVPO saparately yielded only a response to external blue light. Further studies showed also that Nluc-GAVPO fusions driven by a strong CMV promoter had higher activation levels than those driven by a weak SV40 promoter. . Since Nluc-GAVPO with NG configuration had moderate activation levels yet exhibited maximal induction ratio by furimazine, we termed it luminGAVPO (luminescent GAVPO), and the transgene expression system based on luminGAVPO was named the LuminON system.Based on the activation information “OR”-like logic gate is predicted for luminGAVPO-activated transgene expression using furimazine and external blue light as the two inputs. It was also noticed that higher concentrations of furimazine could produce brighter and more sustained luminescence, but Gluc expression slightly decreased when the furimazine concentration is higher than 5 μM. In addition, highly precise control of transgene expression was achieved by combining the furimazine concentration and light irradiance. Another assumption was made, that induction by furimazine multiple times could increase the luminGAVPOmediated activation levels, considering that Nluc is resistant to autoinhibition by its catalytic byproducts. The results showed that Gluc expression increased with increasing round of induction by furimazine.<br /> <br /> Pulsatile activation of transgene expression mediated by luminGAVPO.<br /> Furimazine-induced transgene expression by luminGAVPO would display a pulsatile profile ( activation followed by inactivation). After furimazine induction, high levels of Gluc mRNA and protein production were observed. The results showed that the Gluc synthesis reached a maximum m at ~4–6 h after furimazine induction and then sharply decreased. The medium has also been changed to remove the residual furimazine at 4 h after induction. Therefore, it can be seen that the majority of the furimazine had been consumed within 4 h after induction. It can be concluded that the pulse amplitude and duration of transgene expression were highly dependent on the dose of furimazine. Lower doses of furimazine exhibited shorter durations and smaller amplitudes of pulsatile activation. <br /> <br /> Pulsatile activation of transgene expression in mice.<br /> These results in vitro needed to be proved in vivo. Therefore, luminGAVPO and mCherry reporter plasmids were transfected into the livers of mice.[2] This has been done using a hydrodynamic procedure, which is a rapid injection of a large volume of solution into a vasculature to facilitate substance transfer into parenchyma cells. [3]<br /> Mice were administrated furimazine via tail intravenous (IV), intraperitoneal (IP) or intragastric. mCherry fluorescence was observed in the livers of these mice. After the measured time, the results showed that Gluc expression reached a maximum level within 4 h after furimazine induction and decreased after that. It can be concluded that the half-life of Gluc in the bloodstream is shorter than in the culture medium, and a lower dose of furimazine exhibited shorter durations and smaller amplitudes of Gluc expression in the bloodstream. After all these results demonstrate that the LuminON system can be used for pulsatile and reversible activation of transgene expression in vivo. [2]<br /> <br /> Pulsatile expression of insulin to enhance blood glucose homeostasis in T1D mice.<br /> Insulin is a hormon synthesized by the β-cells of pancreas and is a regulator of metabolism because it maintains glucose homeostasis. [5]<br /> This pulsatile secretion of insulin may match the expression profile mediated by luminGAVPO. HEKFUR-Gluc-P2A-mINS cells were engineered for luminGAVPO-mediated insulin production. [2]<br /> luminGAVPO and UASG-TATA-Gluc-P2A-mINS expression cassettes were integrated into HEK293T cells using the Sleeping Beauty transposon system, which is a promising technology platform for gene transfer in vertebrates. [2,4]<br /> After that clones with the highest Gluc expression were microencapsulated into coherent, semipermeable and immunoprotective alginate-poly-(L-lysine)-alginate beads that allow free diffusion of substances with low molecular weight. T1D mice receiving the microencapsulated cells were treated with 5 mg/kg furimazine, and their blood glucose levels were measured after 3 h. The results showed that , a higher dose of furimazine resulted in more significant restoration of blood glucose and prolonged the maintenance of glucose homeostasis. The results showed the potential in the oral administration of furimazine instead of traditional intramuscular injection of insulin before and after meals. [2]<br /> <br /> == DISCUSSION ==<br /> <br /> Nluc is an ideal luminescence donor to activate LOV protein-based optogenetic systems via BRET, because it has the advantages of high brightness, no requirement of ATP and resistance to autoinhibition by its catalytic byproducts and perfectly matches the absorption spectra of the light-sensitive LOV proteins. In this study BRET-based transgene expression system was used fusing Nluc to a light-switchable transcription factor. Induction by furimazine caused activation of the proximal VVD domain in luminGAVPO, resulting in dimerization of luminGAVPO and binding of luminGAVPO to its cognate promoter, thereby initiating transcription of the target gene. The luminGAVPO dimer gradually dissociates from the promoter, leading to inactivation of transcription, after the furimazine is consumed. That is why this transgene expression is called pulsatile. LuminON system was also used to control the pulsatile and repetitive expression of insulin to enhance blood-glucose homeostasis in T1D mice, which could prevent the hypoglycemia. To conclude, BRET-based optogenetic device developed in this study can be used to precisely control the dynamics of key signaling proteins in a pulsatile fashion both in vitro and in vivo. [2]<br /> <br /> == LITERATURE ==<br /> <br /> [1] Levine JH, Lin Y, Elowitz MB. Functional roles of pulsing in genetic circuits. Science. 2013;342(6163):1193-1200. doi:10.1126/science.1239999 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100686/<br /> <br /> [2] Li, T., Chen, X., Qian, Y. et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat Commun 12, 615 (2021).https://www.nature.com/articles/s41467-021-20913-1#citeas<br /> <br /> [3] Bonamassa B, Hai L, Liu D. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res. 2011;28(4):694-701. doi:10.1007/s11095-010-0338-9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064722/<br /> <br /> [4] Mátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. doi: 10.1038/ng.343. Epub 2009 May 3. PMID: 19412179. https://pubmed.ncbi.nlm.nih.gov/19412179/<br /> <br /> [5] Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25-53. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934755/<br /> <br /> [6]Kobayashi, H., Picard, LP., Schönegge, AM. et al. Bioluminescence resonance energy transfer–based imaging of protein–protein interactions in living cells. Nat Protoc 14, 1084–1107 (2019). https://doi.org/10.1038/s41596-019-0129-7</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena,_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih&diff=18563 2021-04-20T07:46:59Z

<p>Paulahorvat: </p> <hr /> <div>== INTRODUCTION ==<br /> <br /> Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transcription factors and many other regulatory proteins are regulated in a pulsatile fashion, leading to a variety of phenotypic consequences, ranging from stress response to differentiation and signaling.[2]<br /> Pulsing has been observed in many types of proteins. In alternative bacterial sigma factors, but also in mammalian tumor suppressors like p53. Mammalian cells exhibit many pulsatile factors. The stress response pathways mediated by p53, which controls the DNA damage response pulses, just as nuclear factor κB which is involved in immune responses. [1] Iti s still unknown what functional capabilities can this pulsatile dynamic regulation provide for cells. Chemical inducers were used to dynamically regulate transgene expression. Slow degradation and metabolisation of chemical inducers has been shown as not so good application for controlling the dynamics of regulators in most pulsing systems. Therefore, more precise light-inducible systems have been developed that allow control of gene expression. Pulsatile dynamic regulation of regulators is achieved. The limitation that occurs is supplying adequate light intensity to activate these optogenetic systems. To solve this problem luminiscent opsins were developed ( directly coupling bioluminescent proteins to conventional light-sensitive opsins). BRET or bioluminescence resonance energy transfer is how luciferase substrates create bioluminiscence and activate opsins. After some time inactivation of opsins occurs and they regulate the cell activity in a pulsatile fashion. That is where the idea for transgene expression system that allows pulsatile regulation of gene expression both in vitro and in vivo came from. [2]<br /> Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. [6]<br /> <br /> == EXPERIMENT ==<br /> <br /> In this experiment the development of the luminGAVPO, a luminsecent transcription factor is explained. LuminGAVPO enables pulsatile and quantitative activation of transgene expression in both mammalian cells and animals in a furimazine- or light irradiance-dependent manner. By controlling blood glucose homeostasis in type 1 diabetic mice its usefulness is tested. [2]<br /> <br /> == RESULTS ==<br /> <br /> Design and construction of the LuminON transgene expression system.<br /> NanoLuc (Nluc) is a engineered luciferase from a deep sea shrimp, which is stable, bright, small and with sustained luminescence. It is used as luminiscence donor ( produces blue light) to activate LOV-based (The light-oxygen-voltage proteins) optogenetic modules. Nluc was fused to the N terminus of the mCherry-LOV2 fusion protein. When a protein was coexpressed with a mitochondria-anchored mVenus-Zdk protein, both mCherry and mVenus fluorescence accumulated at mitochondria under dark conditions. But the mCherry fluorescence readily diffused to the cytosol within 5 seconds after the addition of furimazine or exposure to external blue ligh. Blue light-inducible transgene expression systems based on Vivid were developed in which transcription factors bind to the promoter region ( to activate or repress gene transcription upon blue light exposure). The next step was to create a luminiscent transcription factor of a chimeric protein that consists of Nluc and a light-switchable transcription factor, GAVPO. Nluc-GAVPO fusions activated Gluc expression upon incubation with furimazine (FZ) or illumination with external blue light. On the other hand expression of Nluc and GAVPO saparately yielded only a response to external blue light. Further studies showed also that Nluc-GAVPO fusions driven by a strong CMV promoter had higher activation levels than those driven by a weak SV40 promoter. . Since Nluc-GAVPO with NG configuration had moderate activation levels yet exhibited maximal induction ratio by furimazine, we termed it luminGAVPO (luminescent GAVPO), and the transgene expression system based on luminGAVPO was named the LuminON system.Based on the activation information “OR”-like logic gate is predicted for luminGAVPO-activated transgene expression using furimazine and external blue light as the two inputs. It was also noticed that higher concentrations of furimazine could produce brighter and more sustained luminescence, but Gluc expression slightly decreased when the furimazine concentration is higher than 5 μM. In addition, highly precise control of transgene expression was achieved by combining the furimazine concentration and light irradiance. Another assumption was made, that induction by furimazine multiple times could increase the luminGAVPOmediated activation levels, considering that Nluc is resistant to autoinhibition by its catalytic byproducts. The results showed that Gluc expression increased with increasing round of induction by furimazine.<br /> <br /> Pulsatile activation of transgene expression mediated by luminGAVPO.<br /> Furimazine-induced transgene expression by luminGAVPO would display a pulsatile profile ( activation followed by inactivation). After furimazine induction, high levels of Gluc mRNA and protein production were observed. The results showed that the Gluc synthesis reached a maximum m at ~4–6 h after furimazine induction and then sharply decreased. The medium has also been changed to remove the residual furimazine at 4 h after induction. Therefore, it can be seen that the majority of the furimazine had been consumed within 4 h after induction. It can be concluded that the pulse amplitude and duration of transgene expression were highly dependent on the dose of furimazine. Lower doses of furimazine exhibited shorter durations and smaller amplitudes of pulsatile activation. <br /> <br /> Pulsatile activation of transgene expression in mice.<br /> These results in vitro needed to be proved in vivo. Therefore, luminGAVPO and mCherry reporter plasmids were transfected into the livers of mice.[2] This has been done using a hydrodynamic procedure, which is a rapid injection of a large volume of solution into a vasculature to facilitate substance transfer into parenchyma cells. [3]<br /> Mice were administrated furimazine via tail intravenous (IV), intraperitoneal (IP) or intragastric. mCherry fluorescence was observed in the livers of these mice. After the measured time, the results showed that Gluc expression reached a maximum level within 4 h after furimazine induction and decreased after that. It can be concluded that the half-life of Gluc in the bloodstream is shorter than in the culture medium, and a lower dose of furimazine exhibited shorter durations and smaller amplitudes of Gluc expression in the bloodstream. After all these results demonstrate that the LuminON system can be used for pulsatile and reversible activation of transgene expression in vivo. [2]<br /> <br /> Pulsatile expression of insulin to enhance blood glucose homeostasis in T1D mice.<br /> Insulin is a hormon synthesized by the β-cells of pancreas and is a regulator of metabolism because it maintains glucose homeostasis. [5]<br /> This pulsatile secretion of insulin may match the expression profile mediated by luminGAVPO. HEKFUR-Gluc-P2A-mINS cells were engineered for luminGAVPO-mediated insulin production. [2]<br /> luminGAVPO and UASG-TATA-Gluc-P2A-mINS expression cassettes were integrated into HEK293T cells using the Sleeping Beauty transposon system, which is a promising technology platform for gene transfer in vertebrates. [2,4]<br /> After that clones with the highest Gluc expression were microencapsulated into coherent, semipermeable and immunoprotective alginate-poly-(L-lysine)-alginate beads that allow free diffusion of substances with low molecular weight. T1D mice receiving the microencapsulated cells were treated with 5 mg/kg furimazine, and their blood glucose levels were measured after 3 h. The results showed that , a higher dose of furimazine resulted in more significant restoration of blood glucose and prolonged the maintenance of glucose homeostasis. The results showed the potential in the oral administration of furimazine instead of traditional intramuscular injection of insulin before and after meals. [2]<br /> <br /> == DISCUSSION ==<br /> <br /> Nluc is an ideal luminescence donor to activate LOV protein-based optogenetic systems via BRET, because it has the advantages of high brightness, no requirement of ATP and resistance to autoinhibition by its catalytic byproducts and perfectly matches the absorption spectra of the light-sensitive LOV proteins. In this study BRET-based transgene expression system was used fusing Nluc to a light-switchable transcription factor. Induction by furimazine caused activation of the proximal VVD domain in luminGAVPO, resulting in dimerization of luminGAVPO and binding of luminGAVPO to its cognate promoter, thereby initiating transcription of the target gene. The luminGAVPO dimer gradually dissociates from the promoter, leading to inactivation of transcription, after the furimazine is consumed. That is why this transgene expression is called pulsatile. LuminON system was also used to control the pulsatile and repetitive expression of insulin to enhance blood-glucose homeostasis in T1D mice, which could prevent the hypoglycemia. To conclude, BRET-based optogenetic device developed in this study can be used to precisely control the dynamics of key signaling proteins in a pulsatile fashion both in vitro and in vivo. [2]<br /> <br /> == LITERATURE ==<br /> <br /> [1] Levine JH, Lin Y, Elowitz MB. Functional roles of pulsing in genetic circuits. Science. 2013;342(6163):1193-1200. doi:10.1126/science.1239999 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100686/<br /> <br /> [2] Li, T., Chen, X., Qian, Y. et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat Commun 12, 615 (2021).https://www.nature.com/articles/s41467-021-20913-1#citeas<br /> <br /> [3] Bonamassa B, Hai L, Liu D. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res. 2011;28(4):694-701. doi:10.1007/s11095-010-0338-9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064722/<br /> <br /> [4] Mátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. doi: 10.1038/ng.343. Epub 2009 May 3. PMID: 19412179. https://pubmed.ncbi.nlm.nih.gov/19412179/<br /> <br /> [5] Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25-53. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934755/<br /> <br /> [6]Kobayashi, H., Picard, LP., Schönegge, AM. et al. Bioluminescence resonance energy transfer–based imaging of protein–protein interactions in living cells. Nat Protoc 14, 1084–1107 (2019). https://doi.org/10.1038/s41596-019-0129-7<br /> https://www.nature.com/articles/s41596-019-0129-7#citeas</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena,_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih&diff=18562 2021-04-20T07:44:07Z

<p>Paulahorvat: </p> <hr /> <div>== INTRODUCTION ==<br /> <br /> Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transcription factors and many other regulatory proteins are regulated in a pulsatile fashion, leading to a variety of phenotypic consequences, ranging from stress response to differentiation and signaling.[2]<br /> Pulsing has been observed in many types of proteins. In alternative bacterial sigma factors, but also in mammalian tumor suppressors like p53. Mammalian cells exhibit many pulsatile factors. The stress response pathways mediated by p53, which controls the DNA damage response pulses, just as nuclear factor κB which is involved in immune responses. [1] Iti s still unknown what functional capabilities can this pulsatile dynamic regulation provide for cells. Chemical inducers were used to dynamically regulate transgene expression. Slow degradation and metabolisation of chemical inducers has been shown as not so good application for controlling the dynamics of regulators in most pulsing systems. Therefore, more precise light-inducible systems have been developed that allow control of gene expression. Pulsatile dynamic regulation of regulators is achieved. The limitation that occurs is supplying adequate light intensity to activate these optogenetic systems. To solve this problem luminiscent opsins were developed ( directly coupling bioluminescent proteins to conventional light-sensitive opsins). BRET or bioluminescence resonance energy transfer is how luciferase substrates create bioluminiscence and activate opsins. After some time inactivation of opsins occurs and they regulate the cell activity in a pulsatile fashion. That is where the idea for transgene expression system that allows pulsatile regulation of gene expression both in vitro and in vivo came from. [2]<br /> <br /> == EXPERIMENT ==<br /> <br /> In this experiment the development of the luminGAVPO, a luminsecent transcription factor is explained. LuminGAVPO enables pulsatile and quantitative activation of transgene expression in both mammalian cells and animals in a furimazine- or light irradiance-dependent manner. By controlling blood glucose homeostasis in type 1 diabetic mice its usefulness is tested. [2]<br /> <br /> == RESULTS ==<br /> <br /> Design and construction of the LuminON transgene expression system.<br /> NanoLuc (Nluc) is a engineered luciferase from a deep sea shrimp, which is stable, bright, small and with sustained luminescence. It is used as luminiscence donor ( produces blue light) to activate LOV-based (The light-oxygen-voltage proteins) optogenetic modules. Nluc was fused to the N terminus of the mCherry-LOV2 fusion protein. When a protein was coexpressed with a mitochondria-anchored mVenus-Zdk protein, both mCherry and mVenus fluorescence accumulated at mitochondria under dark conditions. But the mCherry fluorescence readily diffused to the cytosol within 5 seconds after the addition of furimazine or exposure to external blue ligh. Blue light-inducible transgene expression systems based on Vivid were developed in which transcription factors bind to the promoter region ( to activate or repress gene transcription upon blue light exposure). The next step was to create a luminiscent transcription factor of a chimeric protein that consists of Nluc and a light-switchable transcription factor, GAVPO. Nluc-GAVPO fusions activated Gluc expression upon incubation with furimazine (FZ) or illumination with external blue light. On the other hand expression of Nluc and GAVPO saparately yielded only a response to external blue light. Further studies showed also that Nluc-GAVPO fusions driven by a strong CMV promoter had higher activation levels than those driven by a weak SV40 promoter. . Since Nluc-GAVPO with NG configuration had moderate activation levels yet exhibited maximal induction ratio by furimazine, we termed it luminGAVPO (luminescent GAVPO), and the transgene expression system based on luminGAVPO was named the LuminON system.Based on the activation information “OR”-like logic gate is predicted for luminGAVPO-activated transgene expression using furimazine and external blue light as the two inputs. It was also noticed that higher concentrations of furimazine could produce brighter and more sustained luminescence, but Gluc expression slightly decreased when the furimazine concentration is higher than 5 μM. In addition, highly precise control of transgene expression was achieved by combining the furimazine concentration and light irradiance. Another assumption was made, that induction by furimazine multiple times could increase the luminGAVPOmediated activation levels, considering that Nluc is resistant to autoinhibition by its catalytic byproducts. The results showed that Gluc expression increased with increasing round of induction by furimazine.<br /> <br /> Pulsatile activation of transgene expression mediated by luminGAVPO.<br /> Furimazine-induced transgene expression by luminGAVPO would display a pulsatile profile ( activation followed by inactivation). After furimazine induction, high levels of Gluc mRNA and protein production were observed. The results showed that the Gluc synthesis reached a maximum m at ~4–6 h after furimazine induction and then sharply decreased. The medium has also been changed to remove the residual furimazine at 4 h after induction. Therefore, it can be seen that the majority of the furimazine had been consumed within 4 h after induction. It can be concluded that the pulse amplitude and duration of transgene expression were highly dependent on the dose of furimazine. Lower doses of furimazine exhibited shorter durations and smaller amplitudes of pulsatile activation. <br /> <br /> Pulsatile activation of transgene expression in mice.<br /> These results in vitro needed to be proved in vivo. Therefore, luminGAVPO and mCherry reporter plasmids were transfected into the livers of mice.[2] This has been done using a hydrodynamic procedure, which is a rapid injection of a large volume of solution into a vasculature to facilitate substance transfer into parenchyma cells. [3]<br /> Mice were administrated furimazine via tail intravenous (IV), intraperitoneal (IP) or intragastric. mCherry fluorescence was observed in the livers of these mice. After the measured time, the results showed that Gluc expression reached a maximum level within 4 h after furimazine induction and decreased after that. It can be concluded that the half-life of Gluc in the bloodstream is shorter than in the culture medium, and a lower dose of furimazine exhibited shorter durations and smaller amplitudes of Gluc expression in the bloodstream. After all these results demonstrate that the LuminON system can be used for pulsatile and reversible activation of transgene expression in vivo. [2]<br /> <br /> Pulsatile expression of insulin to enhance blood glucose homeostasis in T1D mice.<br /> Insulin is a hormon synthesized by the β-cells of pancreas and is a regulator of metabolism because it maintains glucose homeostasis. [5]<br /> This pulsatile secretion of insulin may match the expression profile mediated by luminGAVPO. HEKFUR-Gluc-P2A-mINS cells were engineered for luminGAVPO-mediated insulin production. [2]<br /> luminGAVPO and UASG-TATA-Gluc-P2A-mINS expression cassettes were integrated into HEK293T cells using the Sleeping Beauty transposon system, which is a promising technology platform for gene transfer in vertebrates. [2,4]<br /> After that clones with the highest Gluc expression were microencapsulated into coherent, semipermeable and immunoprotective alginate-poly-(L-lysine)-alginate beads that allow free diffusion of substances with low molecular weight. T1D mice receiving the microencapsulated cells were treated with 5 mg/kg furimazine, and their blood glucose levels were measured after 3 h. The results showed that , a higher dose of furimazine resulted in more significant restoration of blood glucose and prolonged the maintenance of glucose homeostasis. The results showed the potential in the oral administration of furimazine instead of traditional intramuscular injection of insulin before and after meals. [2]<br /> <br /> == DISCUSSION ==<br /> <br /> Nluc is an ideal luminescence donor to activate LOV protein-based optogenetic systems via BRET, because it has the advantages of high brightness, no requirement of ATP and resistance to autoinhibition by its catalytic byproducts and perfectly matches the absorption spectra of the light-sensitive LOV proteins. In this study BRET-based transgene expression system was used fusing Nluc to a light-switchable transcription factor. Induction by furimazine caused activation of the proximal VVD domain in luminGAVPO, resulting in dimerization of luminGAVPO and binding of luminGAVPO to its cognate promoter, thereby initiating transcription of the target gene. The luminGAVPO dimer gradually dissociates from the promoter, leading to inactivation of transcription, after the furimazine is consumed. That is why this transgene expression is called pulsatile. LuminON system was also used to control the pulsatile and repetitive expression of insulin to enhance blood-glucose homeostasis in T1D mice, which could prevent the hypoglycemia. To conclude, BRET-based optogenetic device developed in this study can be used to precisely control the dynamics of key signaling proteins in a pulsatile fashion both in vitro and in vivo. [2]<br /> <br /> == LITERATURE ==<br /> <br /> [1] Levine JH, Lin Y, Elowitz MB. Functional roles of pulsing in genetic circuits. Science. 2013;342(6163):1193-1200. doi:10.1126/science.1239999 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100686/<br /> <br /> [2] Li, T., Chen, X., Qian, Y. et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat Commun 12, 615 (2021).https://www.nature.com/articles/s41467-021-20913-1#citeas<br /> <br /> [3] Bonamassa B, Hai L, Liu D. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res. 2011;28(4):694-701. doi:10.1007/s11095-010-0338-9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064722/<br /> <br /> [4] Mátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. doi: 10.1038/ng.343. Epub 2009 May 3. PMID: 19412179. https://pubmed.ncbi.nlm.nih.gov/19412179/<br /> <br /> [5] Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25-53. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934755/</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena,_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih&diff=18561 2021-04-20T07:42:26Z

<p>Paulahorvat: </p> <hr /> <div>== INTRODUCTION ==<br /> <br /> Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transcription factors and many other regulatory proteins are regulated in a pulsatile fashion, leading to a variety of phenotypic consequences, ranging from stress response to differentiation and signaling.[2]<br /> Pulsing has been observed in many types of proteins. In alternative bacterial sigma factors, but also in mammalian tumor suppressors like p53. Mammalian cells exhibit many pulsatile factors. The stress response pathways mediated by p53, which controls the DNA damage response pulses, just as nuclear factor κB which is involved in immune responses. [1] Iti s still unknown what functional capabilities can this pulsatile dynamic regulation provide for cells. Chemical inducers were used to dynamically regulate transgene expression. Slow degradation and metabolisation of chemical inducers has been shown as not so good application for controlling the dynamics of regulators in most pulsing systems. Therefore, more precise light-inducible systems have been developed that allow control of gene expression. Pulsatile dynamic regulation of regulators is achieved. The limitation that occurs is supplying adequate light intensity to activate these optogenetic systems. To solve this problem luminiscent opsins were developed ( directly coupling bioluminescent proteins to conventional light-sensitive opsins). BRET or bioluminescence resonance energy transfer is how luciferase substrates create bioluminiscence and activate opsins. After some time inactivation of opsins occurs and they regulate the cell activity in a pulsatile fashion. That is where the idea for transgene expression system that allows pulsatile regulation of gene expression both in vitro and in vivo came from. [2]<br /> <br /> == EXPERIMENT ==<br /> <br /> In this experiment the development of the luminGAVPO, a luminsecent transcription factor is explained. LuminGAVPO enables pulsatile and quantitative activation of transgene expression in both mammalian cells and animals in a furimazine- or light irradiance-dependent manner. By controlling blood glucose homeostasis in type 1 diabetic mice its usefulness is tested. [2]<br /> <br /> == RESULTS ==<br /> <br /> Design and construction of the LuminON transgene expression system.<br /> NanoLuc (Nluc) is a engineered luciferase from a deep sea shrimp, which is stable, bright, small and with sustained luminescence. It is used as luminiscence donor ( produces blue light) to activate LOV-based (The light-oxygen-voltage proteins) optogenetic modules. Nluc was fused to the N terminus of the mCherry-LOV2 fusion protein. When a protein was coexpressed with a mitochondria-anchored mVenus-Zdk protein, both mCherry and mVenus fluorescence accumulated at mitochondria under dark conditions. But the mCherry fluorescence readily diffused to the cytosol within 5 seconds after the addition of furimazine or exposure to external blue ligh. Blue light-inducible transgene expression systems based on Vivid were developed in which transcription factors bind to the promoter region ( to activate or repress gene transcription upon blue light exposure). The next step was to create a luminiscent transcription factor of a chimeric protein that consists of Nluc and a light-switchable transcription factor, GAVPO. Nluc-GAVPO fusions activated Gluc expression upon incubation with furimazine (FZ) or illumination with external blue light. On the other hand expression of Nluc and GAVPO saparately yielded only a response to external blue light. Further studies showed also that Nluc-GAVPO fusions driven by a strong CMV promoter had higher activation levels than those driven by a weak SV40 promoter. . Since Nluc-GAVPO with NG configuration had moderate activation levels yet exhibited maximal induction ratio by furimazine, we termed it luminGAVPO (luminescent GAVPO), and the transgene expression system based on luminGAVPO was named the LuminON system.Based on the activation information “OR”-like logic gate is predicted for luminGAVPO-activated transgene expression using furimazine and external blue light as the two inputs. It was also noticed that higher concentrations of furimazine could produce brighter and more sustained luminescence, but Gluc expression slightly decreased when the furimazine concentration is higher than 5 μM. In addition, highly precise control of transgene expression was achieved by combining the furimazine concentration and light irradiance. Another assumption was made, that induction by furimazine multiple times could increase the luminGAVPOmediated activation levels, considering that Nluc is resistant to autoinhibition by its catalytic byproducts. The results showed that Gluc expression increased with increasing round of induction by furimazine.<br /> <br /> Pulsatile activation of transgene expression mediated by luminGAVPO.<br /> Furimazine-induced transgene expression by luminGAVPO would display a pulsatile profile ( activation followed by inactivation). After furimazine induction, high levels of Gluc mRNA and protein production were observed. The results showed that the Gluc synthesis reached a maximum m at ~4–6 h after furimazine induction and then sharply decreased. The medium has also been changed to remove the residual furimazine at 4 h after induction. Therefore, it can be seen that the majority of the furimazine had been consumed within 4 h after induction. It can be concluded that the pulse amplitude and duration of transgene expression were highly dependent on the dose of furimazine. Lower doses of furimazine exhibited shorter durations and smaller amplitudes of pulsatile activation. <br /> <br /> Pulsatile activation of transgene expression in mice.<br /> These results in vitro needed to be proved in vivo. Therefore, luminGAVPO and mCherry reporter plasmids were transfected into the livers of mice.[2] This has been done using a hydrodynamic procedure, which is a rapid injection of a large volume of solution into a vasculature to facilitate substance transfer into parenchyma cells. [3]<br /> Mice were administrated furimazine via tail intravenous (IV), intraperitoneal (IP) or intragastric. mCherry fluorescence was observed in the livers of these mice. After the measured time, the results showed that Gluc expression reached a maximum level within 4 h after furimazine induction and decreased after that. It can be concluded that the half-life of Gluc in the bloodstream is shorter than in the culture medium, and a lower dose of furimazine exhibited shorter durations and smaller amplitudes of Gluc expression in the bloodstream. After all these results demonstrate that the LuminON system can be used for pulsatile and reversible activation of transgene expression in vivo. [2]<br /> <br /> Pulsatile expression of insulin to enhance blood glucose homeostasis in T1D mice.<br /> Insulin is a hormon synthesized by the β-cells of pancreas and is a regulator of metabolism because it maintains glucose homeostasis. [5]<br /> This pulsatile secretion of insulin may match the expression profile mediated by luminGAVPO. HEKFUR-Gluc-P2A-mINS cells were engineered for luminGAVPO-mediated insulin production. [2]<br /> luminGAVPO and UASG-TATA-Gluc-P2A-mINS expression cassettes were integrated into HEK293T cells using the Sleeping Beauty transposon system, which is a promising technology platform for gene transfer in vertebrates. [2,4]<br /> After that clones with the highest Gluc expression were microencapsulated into coherent, semipermeable and immunoprotective alginate-poly-(L-lysine)-alginate beads that allow free diffusion of substances with low molecular weight. T1D mice receiving the microencapsulated cells were treated with 5 mg/kg furimazine, and their blood glucose levels were measured after 3 h. The results showed that , a higher dose of furimazine resulted in more significant restoration of blood glucose and prolonged the maintenance of glucose homeostasis. The results showed the potential in the oral administration of furimazine instead of traditional intramuscular injection of insulin before and after meals. [2]<br /> <br /> == DISCUSSION ==<br /> <br /> Nluc is an ideal luminescence donor to activate LOV protein-based optogenetic systems via BRET, because it has the advantages of high brightness, no requirement of ATP and resistance to autoinhibition by its catalytic byproducts and perfectly matches the absorption spectra of the light-sensitive LOV proteins. In this study BRET-based transgene expression system was used fusing Nluc to a light-switchable transcription factor. Induction by furimazine caused activation of the proximal VVD domain in luminGAVPO, resulting in dimerization of luminGAVPO and binding of luminGAVPO to its cognate promoter, thereby initiating transcription of the target gene. The luminGAVPO dimer gradually dissociates from the promoter, leading to inactivation of transcription, after the furimazine is consumed. That is why this transgene expression is called pulsatile. LuminON system was also used to control the pulsatile and repetitive expression of insulin to enhance blood-glucose homeostasis in T1D mice, which could prevent the hypoglycemia. To conclude, BRET-based optogenetic device developed in this study can be used to precisely control the dynamics of key signaling proteins in a pulsatile fashion both in vitro and in vivo. [2]<br /> <br /> == LITERATURE ==<br /> <br /> [1] Levine JH, Lin Y, Elowitz MB. Functional roles of pulsing in genetic circuits. Science. 2013;342(6163):1193-1200. doi:10.1126/science.1239999 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100686/<br /> [2] Li, T., Chen, X., Qian, Y. et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat Commun 12, 615 (2021).https://www.nature.com/articles/s41467-021-20913-1#citeas<br /> [3] Bonamassa B, Hai L, Liu D. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res. 2011;28(4):694-701. doi:10.1007/s11095-010-0338-9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064722/<br /> [4] Mátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. doi: 10.1038/ng.343. Epub 2009 May 3. PMID: 19412179. https://pubmed.ncbi.nlm.nih.gov/19412179/<br /> [5] Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25-53. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934755/</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena,_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih&diff=18560 2021-04-20T07:39:26Z

<p>Paulahorvat: </p> <hr /> <div>== INTRODUCTION ==<br /> <br /> Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transcription factors and many other regulatory proteins are regulated in a pulsatile fashion, leading to a variety of phenotypic consequences, ranging from stress response to differentiation and signaling.[2]<br /> Pulsing has been observed in many types of proteins. In alternative bacterial sigma factors, but also in mammalian tumor suppressors like p53. Mammalian cells exhibit many pulsatile factors. The stress response pathways mediated by p53, which controls the DNA damage response pulses, just as nuclear factor κB which is involved in immune responses. [1] Iti s still unknown what functional capabilities can this pulsatile dynamic regulation provide for cells. Chemical inducers were used to dynamically regulate transgene expression. Slow degradation and metabolisation of chemical inducers has been shown as not so good application for controlling the dynamics of regulators in most pulsing systems. Therefore, more precise light-inducible systems have been developed that allow control of gene expression. Pulsatile dynamic regulation of regulators is achieved. The limitation that occurs is supplying adequate light intensity to activate these optogenetic systems. To solve this problem luminiscent opsins were developed ( directly coupling bioluminescent proteins to conventional light-sensitive opsins). BRET or bioluminescence resonance energy transfer is how luciferase substrates create bioluminiscence and activate opsins. After some time inactivation of opsins occurs and they regulate the cell activity in a pulsatile fashion. That is where the idea for transgene expression system that allows pulsatile regulation of gene expression both in vitro and in vivo came from. [2]<br /> <br /> == EXPERIMENT ==<br /> <br /> In this experiment the development of the luminGAVPO, a luminsecent transcription factor is explained. LuminGAVPO enables pulsatile and quantitative activation of transgene expression in both mammalian cells and animals in a furimazine- or light irradiance-dependent manner. By controlling blood glucose homeostasis in type 1 diabetic mice its usefulness is tested. [2]<br /> <br /> == RESULTS ==<br /> <br /> Design and construction of the LuminON transgene expression system.<br /> NanoLuc (Nluc) is a engineered luciferase from a deep sea shrimp, which is stable, bright, small and with sustained luminescence. It is used as luminiscence donor ( produces blue light) to activate LOV-based (The light-oxygen-voltage proteins) optogenetic modules. Nluc was fused to the N terminus of the mCherry-LOV2 fusion protein. When a protein was coexpressed with a mitochondria-anchored mVenus-Zdk protein, both mCherry and mVenus fluorescence accumulated at mitochondria under dark conditions. But the mCherry fluorescence readily diffused to the cytosol within 5 seconds after the addition of furimazine or exposure to external blue ligh. Blue light-inducible transgene expression systems based on Vivid were developed in which transcription factors bind to the promoter region ( to activate or repress gene transcription upon blue light exposure). The next step was to create a luminiscent transcription factor of a chimeric protein that consists of Nluc and a light-switchable transcription factor, GAVPO. Nluc-GAVPO fusions activated Gluc expression upon incubation with furimazine (FZ) or illumination with external blue light. On the other hand expression of Nluc and GAVPO saparately yielded only a response to external blue light. Further studies showed also that Nluc-GAVPO fusions driven by a strong CMV promoter had higher activation levels than those driven by a weak SV40 promoter. . Since Nluc-GAVPO with NG configuration had moderate activation levels yet exhibited maximal induction ratio by furimazine, we termed it luminGAVPO (luminescent GAVPO), and the transgene expression system based on luminGAVPO was named the LuminON system.Based on the activation information “OR”-like logic gate is predicted for luminGAVPO-activated transgene expression using furimazine and external blue light as the two inputs. It was also noticed that higher concentrations of furimazine could produce brighter and more sustained luminescence, but Gluc expression slightly decreased when the furimazine concentration is higher than 5 μM. In addition, highly precise control of transgene expression was achieved by combining the furimazine concentration and light irradiance. Another assumption was made, that induction by furimazine multiple times could increase the luminGAVPOmediated activation levels, considering that Nluc is resistant to autoinhibition by its catalytic byproducts. The results showed that Gluc expression increased with increasing round of induction by furimazine.<br /> <br /> Pulsatile activation of transgene expression mediated by luminGAVPO.<br /> Furimazine-induced transgene expression by luminGAVPO would display a pulsatile profile ( activation followed by inactivation). After furimazine induction, high levels of Gluc mRNA and protein production were observed. The results showed that the Gluc synthesis reached a maximum m at ~4–6 h after furimazine induction and then sharply decreased. The medium has also been changed to remove the residual furimazine at 4 h after induction. Therefore, it can be seen that the majority of the furimazine had been consumed within 4 h after induction. It can be concluded that the pulse amplitude and duration of transgene expression were highly dependent on the dose of furimazine. Lower doses of furimazine exhibited shorter durations and smaller amplitudes of pulsatile activation. <br /> <br /> Pulsatile activation of transgene expression in mice.<br /> These results in vitro needed to be proved in vivo. Therefore, luminGAVPO and mCherry reporter plasmids were transfected into the livers of mice.[2] This has been done using a hydrodynamic procedure, which is a rapid injection of a large volume of solution into a vasculature to facilitate substance transfer into parenchyma cells. [3]<br /> Mice were administrated furimazine via tail intravenous (IV), intraperitoneal (IP) or intragastric. mCherry fluorescence was observed in the livers of these mice. After the measured time, the results showed that Gluc expression reached a maximum level within 4 h after furimazine induction and decreased after that. It can be concluded that the half-life of Gluc in the bloodstream is shorter than in the culture medium, and a lower dose of furimazine exhibited shorter durations and smaller amplitudes of Gluc expression in the bloodstream. After all these results demonstrate that the LuminON system can be used for pulsatile and reversible activation of transgene expression in vivo. [2]<br /> <br /> Pulsatile expression of insulin to enhance blood glucose homeostasis in T1D mice.<br /> Insulin is a hormon synthesized by the β-cells of pancreas and is a regulator of metabolism because it maintains glucose homeostasis. [5]<br /> This pulsatile secretion of insulin may match the expression profile mediated by luminGAVPO. HEKFUR-Gluc-P2A-mINS cells were engineered for luminGAVPO-mediated insulin production. [2]<br /> luminGAVPO and UASG-TATA-Gluc-P2A-mINS expression cassettes were integrated into HEK293T cells using the Sleeping Beauty transposon system, which is a promising technology platform for gene transfer in vertebrates. [2,4]<br /> After that clones with the highest Gluc expression were microencapsulated into coherent, semipermeable and immunoprotective alginate-poly-(L-lysine)-alginate beads that allow free diffusion of substances with low molecular weight. T1D mice receiving the microencapsulated cells were treated with 5 mg/kg furimazine, and their blood glucose levels were measured after 3 h. The results showed that , a higher dose of furimazine resulted in more significant restoration of blood glucose and prolonged the maintenance of glucose homeostasis. The results showed the potential in the oral administration of furimazine instead of traditional intramuscular injection of insulin before and after meals. [2]<br /> <br /> == DISCUSSION ==<br /> <br /> Nluc is an ideal luminescence donor to activate LOV protein-based optogenetic systems via BRET, because it has the advantages of high brightness, no requirement of ATP and resistance to autoinhibition by its catalytic byproducts and perfectly matches the absorption spectra of the light-sensitive LOV proteins. In this study BRET-based transgene expression system was used fusing Nluc to a light-switchable transcription factor. Induction by furimazine caused activation of the proximal VVD domain in luminGAVPO, resulting in dimerization of luminGAVPO and binding of luminGAVPO to its cognate promoter, thereby initiating transcription of the target gene. The luminGAVPO dimer gradually dissociates from the promoter, leading to inactivation of transcription, after the furimazine is consumed. That is why this transgene expression is called pulsatile. LuminON system was also used to control the pulsatile and repetitive expression of insulin to enhance blood-glucose homeostasis in T1D mice, which could prevent the hypoglycemia. To conclude, BRET-based optogenetic device developed in this study can be used to precisely control the dynamics of key signaling proteins in a pulsatile fashion both in vitro and in vivo. [2]<br /> <br /> == LITERATURE ==<br /> <br /> [1] Levine JH, Lin Y, Elowitz MB. Functional roles of pulsing in genetic circuits. Science. 2013;342(6163):1193-1200. doi:10.1126/science.1239999<br /> [2] Li, T., Chen, X., Qian, Y. et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat Commun 12, 615 (2021).<br /> [3] Bonamassa B, Hai L, Liu D. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res. 2011;28(4):694-701. doi:10.1007/s11095-010-0338-9<br /> [4] Mátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. doi: 10.1038/ng.343. Epub 2009 May 3. PMID: 19412179.<br /> [5] Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25-53.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Seminarji_SB_2020/21&diff=18559 2021-04-19T22:00:43Z

<p>Paulahorvat: </p> <hr /> <div>V študijskem letu 2020/21 študentje 1. letnika predstavljajo naslednje teme: <br /> <br /> RAZISKOVALNI ČLANKI<br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do izhodiščnega članka na spletu.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/Proizvodnja_1,3-propandiola_iz_različnih_ogljikovodikov_po_nenaravni_poti_preko_3-hidroksipropanojske_kisline Proizvodnja 1,3-propandiola iz različnih ogljikovodikov po nenaravni poti preko 3-hidroksipropanojske kisline] <br /> (Liza Ulčakar) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/Doseganje_asimetri%C4%8Dnosti_in_asimetri%C4%8Dne_delitve_pri_E._coli Doseganje asimetričnosti in asimetrične delitve pri E. coli] (Aljaž Bratina) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/Zmanj%C5%A1ana_procesivnost_ribosomov_v_sistemu_PURE Zmanjšana procesivnost ribosomov v sistemu PURE] (Tina Kolenc Milavec) &lt;br&gt;<br /> 4 [http://wiki.fkkt.uni-lj.si/index.php/Na%C4%8Drtovana_pot_zvijanja_proteinskih_origamijev Načrtovana pot zvijanja proteinskih origamijev] (Anamarija Agnič) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/U%C4%8Dinkovita_svetlobno_inducibilna_Dre_rekombinaza_za_%C4%8Dasovno_in_prostorsko_celi%C4%8Dno_specifi%C4%8Dno_urejanje_genoma_v_mi%C5%A1jih_modelih#VIRI Učinkovita svetlobno inducibilna Dre rekombinaza za časovno in prostorsko celično specifično urejanje genoma v mišjih modelih] (Nika Mikulič Vernik) &lt;br&gt;<br /> 6 [http://wiki.fkkt.uni-lj.si/index.php/Vzpostavitev_termometra_tRNA_za_dolo%C4%8Danje_temperature_optimalne_rasti_mikroorganizmov Vzpostavitev termometra tRNA za določanje temperature optimalne rasti mikroorganizmov] (Urša Štrancar) &lt;br&gt;<br /> 7 [http://wiki.fkkt.uni-lj.si/index.php/Racionalna_zasnova_minimalnih_sinteti%C4%8Dnih_promotorjev_za_rastline#Construction_of_plasmids Racionalna zasnova minimalnih sintetičnih promotorjev za rastline] (Almina Tahirović) &lt;br&gt;<br /> 8 [http://wiki.fkkt.uni-lj.si/index.php/Reverzibilna_toplotna_regulacija_za_bifunkcionalno_dinamično_uravnavanje_izražanja_genov_v_E._coli Reverzibilna toplotna regulacija za bifunkcionalno dinamično uravnavanje izražanja genov v E. coli] (Urška Fajdiga) &lt;br&gt;<br /> 9 [http://wiki.fkkt.uni-lj.si/index.php/Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena%2C_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih Sintetična optogenetska naprava na osnovi BRET za pulzirajočo ekspresijo transgena, ki omogoča glukozno homeostazo pri miših] (Paula Horvat) &lt;br&gt;<br /> <br /> NAGRAJENI ŠTUDENTSKI PROJEKTI <br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do wiki strani študentske ekipe, katere projekt opisujete.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/RESHAPE_-_spreminjanje_morfologije_nitastih_gliv RESHAPE - spreminjanje morfologije nitastih gliv] <br /> (Špela Supej) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/The_Chlamy_Cleaner:_razgradnja_pesticida_z_zeleno_algo The Chlamy Cleaner: razgradnja pesticida z zeleno algo] (Doroteja Armič) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/TheraPUFA:_nazalni_probiotik_proti_okužbam_in_vnetjem TheraPUFA- nazalni probiotik proti okužbam in vnetjem] (Barbara Slapnik) &lt;br&gt;<br /> 4 [[S-POP: Modularni biosenzor za zaznavanje obstojnih organskih onesnaževal v okoljskih vodah]] (Tadej Medved) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/MARS-magnetni_sistem_za_recikliranje_ATP MARS-magnetni sistem za recikliranje ATP] (David Miškić) &lt;br&gt;<br /> 6 [[B.O.T.: Bakterijska oscilacijska terapija za zdravljenje kolorektalnega raka]] (Neža Pavko) &lt;br&gt;<br /> 7 [[iGEMINI: Kvasovke kot prehransko dopolnilo v vesolju]] (Klementina Polanec) &lt;br&gt;<br /> 8 [[Antea-Glyphosate: Detekcija in razgradnja glifosata]] (Jernej Imperl) &lt;br&gt;<br /> ----<br /> <br /> Povzetki v slovenščini naj imajo 1200-1500 besed (viri v to vsoto ne štejejo). Povzetek je treba objaviti dva dni pred predstavitvijo do polnoči (za seminarje v sredo torej v ponedeljek). Predstavitev seminarja naj bo dolga 15 minut (13-17). Sledila bo razprava, ki praviloma ne bo daljša od 5 minut.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Seminarji_SB_2020/21&diff=18558 2021-04-19T22:00:33Z

<p>Paulahorvat: </p> <hr /> <div>V študijskem letu 2020/21 študentje 1. letnika predstavljajo naslednje teme: <br /> <br /> RAZISKOVALNI ČLANKI<br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do izhodiščnega članka na spletu.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/Proizvodnja_1,3-propandiola_iz_različnih_ogljikovodikov_po_nenaravni_poti_preko_3-hidroksipropanojske_kisline Proizvodnja 1,3-propandiola iz različnih ogljikovodikov po nenaravni poti preko 3-hidroksipropanojske kisline] <br /> (Liza Ulčakar) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/Doseganje_asimetri%C4%8Dnosti_in_asimetri%C4%8Dne_delitve_pri_E._coli Doseganje asimetričnosti in asimetrične delitve pri E. coli] (Aljaž Bratina) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/Zmanj%C5%A1ana_procesivnost_ribosomov_v_sistemu_PURE Zmanjšana procesivnost ribosomov v sistemu PURE] (Tina Kolenc Milavec) &lt;br&gt;<br /> 4 [http://wiki.fkkt.uni-lj.si/index.php/Na%C4%8Drtovana_pot_zvijanja_proteinskih_origamijev Načrtovana pot zvijanja proteinskih origamijev] (Anamarija Agnič) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/U%C4%8Dinkovita_svetlobno_inducibilna_Dre_rekombinaza_za_%C4%8Dasovno_in_prostorsko_celi%C4%8Dno_specifi%C4%8Dno_urejanje_genoma_v_mi%C5%A1jih_modelih#VIRI Učinkovita svetlobno inducibilna Dre rekombinaza za časovno in prostorsko celično specifično urejanje genoma v mišjih modelih] (Nika Mikulič Vernik) &lt;br&gt;<br /> 6 [http://wiki.fkkt.uni-lj.si/index.php/Vzpostavitev_termometra_tRNA_za_dolo%C4%8Danje_temperature_optimalne_rasti_mikroorganizmov Vzpostavitev termometra tRNA za določanje temperature optimalne rasti mikroorganizmov] (Urša Štrancar) &lt;br&gt;<br /> 7 [http://wiki.fkkt.uni-lj.si/index.php/Racionalna_zasnova_minimalnih_sinteti%C4%8Dnih_promotorjev_za_rastline#Construction_of_plasmids Racionalna zasnova minimalnih sintetičnih promotorjev za rastline] (Almina Tahirović) &lt;br&gt;<br /> 8 [http://wiki.fkkt.uni-lj.si/index.php/Reverzibilna_toplotna_regulacija_za_bifunkcionalno_dinamično_uravnavanje_izražanja_genov_v_E._coli Reverzibilna toplotna regulacija za bifunkcionalno dinamično uravnavanje izražanja genov v E. coli] (Urška Fajdiga) &lt;br&gt;<br /> 9[http://wiki.fkkt.uni-lj.si/index.php/Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena%2C_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih Sintetična optogenetska naprava na osnovi BRET za pulzirajočo ekspresijo transgena, ki omogoča glukozno homeostazo pri miših] (Paula Horvat) &lt;br&gt;<br /> <br /> NAGRAJENI ŠTUDENTSKI PROJEKTI <br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do wiki strani študentske ekipe, katere projekt opisujete.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/RESHAPE_-_spreminjanje_morfologije_nitastih_gliv RESHAPE - spreminjanje morfologije nitastih gliv] <br /> (Špela Supej) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/The_Chlamy_Cleaner:_razgradnja_pesticida_z_zeleno_algo The Chlamy Cleaner: razgradnja pesticida z zeleno algo] (Doroteja Armič) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/TheraPUFA:_nazalni_probiotik_proti_okužbam_in_vnetjem TheraPUFA- nazalni probiotik proti okužbam in vnetjem] (Barbara Slapnik) &lt;br&gt;<br /> 4 [[S-POP: Modularni biosenzor za zaznavanje obstojnih organskih onesnaževal v okoljskih vodah]] (Tadej Medved) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/MARS-magnetni_sistem_za_recikliranje_ATP MARS-magnetni sistem za recikliranje ATP] (David Miškić) &lt;br&gt;<br /> 6 [[B.O.T.: Bakterijska oscilacijska terapija za zdravljenje kolorektalnega raka]] (Neža Pavko) &lt;br&gt;<br /> 7 [[iGEMINI: Kvasovke kot prehransko dopolnilo v vesolju]] (Klementina Polanec) &lt;br&gt;<br /> 8 [[Antea-Glyphosate: Detekcija in razgradnja glifosata]] (Jernej Imperl) &lt;br&gt;<br /> ----<br /> <br /> Povzetki v slovenščini naj imajo 1200-1500 besed (viri v to vsoto ne štejejo). Povzetek je treba objaviti dva dni pred predstavitvijo do polnoči (za seminarje v sredo torej v ponedeljek). Predstavitev seminarja naj bo dolga 15 minut (13-17). Sledila bo razprava, ki praviloma ne bo daljša od 5 minut.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Seminarji_SB_2020/21&diff=18557 2021-04-19T21:57:31Z

<p>Paulahorvat: </p> <hr /> <div>V študijskem letu 2020/21 študentje 1. letnika predstavljajo naslednje teme: <br /> <br /> RAZISKOVALNI ČLANKI<br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do izhodiščnega članka na spletu.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/Proizvodnja_1,3-propandiola_iz_različnih_ogljikovodikov_po_nenaravni_poti_preko_3-hidroksipropanojske_kisline Proizvodnja 1,3-propandiola iz različnih ogljikovodikov po nenaravni poti preko 3-hidroksipropanojske kisline] <br /> (Liza Ulčakar) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/Doseganje_asimetri%C4%8Dnosti_in_asimetri%C4%8Dne_delitve_pri_E._coli Doseganje asimetričnosti in asimetrične delitve pri E. coli] (Aljaž Bratina) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/Zmanj%C5%A1ana_procesivnost_ribosomov_v_sistemu_PURE Zmanjšana procesivnost ribosomov v sistemu PURE] (Tina Kolenc Milavec) &lt;br&gt;<br /> 4 [http://wiki.fkkt.uni-lj.si/index.php/Na%C4%8Drtovana_pot_zvijanja_proteinskih_origamijev Načrtovana pot zvijanja proteinskih origamijev] (Anamarija Agnič) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/U%C4%8Dinkovita_svetlobno_inducibilna_Dre_rekombinaza_za_%C4%8Dasovno_in_prostorsko_celi%C4%8Dno_specifi%C4%8Dno_urejanje_genoma_v_mi%C5%A1jih_modelih#VIRI Učinkovita svetlobno inducibilna Dre rekombinaza za časovno in prostorsko celično specifično urejanje genoma v mišjih modelih] (Nika Mikulič Vernik) &lt;br&gt;<br /> 6 [http://wiki.fkkt.uni-lj.si/index.php/Vzpostavitev_termometra_tRNA_za_dolo%C4%8Danje_temperature_optimalne_rasti_mikroorganizmov Vzpostavitev termometra tRNA za določanje temperature optimalne rasti mikroorganizmov] (Urša Štrancar) &lt;br&gt;<br /> 7 [http://wiki.fkkt.uni-lj.si/index.php/Racionalna_zasnova_minimalnih_sinteti%C4%8Dnih_promotorjev_za_rastline#Construction_of_plasmids Racionalna zasnova minimalnih sintetičnih promotorjev za rastline] (Almina Tahirović) &lt;br&gt;<br /> 8 [http://wiki.fkkt.uni-lj.si/index.php/Reverzibilna_toplotna_regulacija_za_bifunkcionalno_dinamično_uravnavanje_izražanja_genov_v_E._coli Reverzibilna toplotna regulacija za bifunkcionalno dinamično uravnavanje izražanja genov v E. coli] (Urška Fajdiga) &lt;br&gt;<br /> 9[http://wiki.fkkt.uni-lj.si/index.php/Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena%2C_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih] (Paula Horvat) &lt;br&gt;<br /> <br /> NAGRAJENI ŠTUDENTSKI PROJEKTI <br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do wiki strani študentske ekipe, katere projekt opisujete.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/RESHAPE_-_spreminjanje_morfologije_nitastih_gliv RESHAPE - spreminjanje morfologije nitastih gliv] <br /> (Špela Supej) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/The_Chlamy_Cleaner:_razgradnja_pesticida_z_zeleno_algo The Chlamy Cleaner: razgradnja pesticida z zeleno algo] (Doroteja Armič) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/TheraPUFA:_nazalni_probiotik_proti_okužbam_in_vnetjem TheraPUFA- nazalni probiotik proti okužbam in vnetjem] (Barbara Slapnik) &lt;br&gt;<br /> 4 [[S-POP: Modularni biosenzor za zaznavanje obstojnih organskih onesnaževal v okoljskih vodah]] (Tadej Medved) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/MARS-magnetni_sistem_za_recikliranje_ATP MARS-magnetni sistem za recikliranje ATP] (David Miškić) &lt;br&gt;<br /> 6 [[B.O.T.: Bakterijska oscilacijska terapija za zdravljenje kolorektalnega raka]] (Neža Pavko) &lt;br&gt;<br /> 7 [[iGEMINI: Kvasovke kot prehransko dopolnilo v vesolju]] (Klementina Polanec) &lt;br&gt;<br /> 8 [[Antea-Glyphosate: Detekcija in razgradnja glifosata]] (Jernej Imperl) &lt;br&gt;<br /> ----<br /> <br /> Povzetki v slovenščini naj imajo 1200-1500 besed (viri v to vsoto ne štejejo). Povzetek je treba objaviti dva dni pred predstavitvijo do polnoči (za seminarje v sredo torej v ponedeljek). Predstavitev seminarja naj bo dolga 15 minut (13-17). Sledila bo razprava, ki praviloma ne bo daljša od 5 minut.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Seminarji_SB_2020/21&diff=18556 2021-04-19T21:55:33Z

<p>Paulahorvat: </p> <hr /> <div>V študijskem letu 2020/21 študentje 1. letnika predstavljajo naslednje teme: <br /> <br /> RAZISKOVALNI ČLANKI<br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do izhodiščnega članka na spletu.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/Proizvodnja_1,3-propandiola_iz_različnih_ogljikovodikov_po_nenaravni_poti_preko_3-hidroksipropanojske_kisline Proizvodnja 1,3-propandiola iz različnih ogljikovodikov po nenaravni poti preko 3-hidroksipropanojske kisline] <br /> (Liza Ulčakar) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/Doseganje_asimetri%C4%8Dnosti_in_asimetri%C4%8Dne_delitve_pri_E._coli Doseganje asimetričnosti in asimetrične delitve pri E. coli] (Aljaž Bratina) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/Zmanj%C5%A1ana_procesivnost_ribosomov_v_sistemu_PURE Zmanjšana procesivnost ribosomov v sistemu PURE] (Tina Kolenc Milavec) &lt;br&gt;<br /> 4 [http://wiki.fkkt.uni-lj.si/index.php/Na%C4%8Drtovana_pot_zvijanja_proteinskih_origamijev Načrtovana pot zvijanja proteinskih origamijev] (Anamarija Agnič) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/U%C4%8Dinkovita_svetlobno_inducibilna_Dre_rekombinaza_za_%C4%8Dasovno_in_prostorsko_celi%C4%8Dno_specifi%C4%8Dno_urejanje_genoma_v_mi%C5%A1jih_modelih#VIRI Učinkovita svetlobno inducibilna Dre rekombinaza za časovno in prostorsko celično specifično urejanje genoma v mišjih modelih] (Nika Mikulič Vernik) &lt;br&gt;<br /> 6 [http://wiki.fkkt.uni-lj.si/index.php/Vzpostavitev_termometra_tRNA_za_dolo%C4%8Danje_temperature_optimalne_rasti_mikroorganizmov Vzpostavitev termometra tRNA za določanje temperature optimalne rasti mikroorganizmov] (Urša Štrancar) &lt;br&gt;<br /> 7 [http://wiki.fkkt.uni-lj.si/index.php/Racionalna_zasnova_minimalnih_sinteti%C4%8Dnih_promotorjev_za_rastline#Construction_of_plasmids Racionalna zasnova minimalnih sintetičnih promotorjev za rastline] (Almina Tahirović) &lt;br&gt;<br /> 8 [http://wiki.fkkt.uni-lj.si/index.php/Reverzibilna_toplotna_regulacija_za_bifunkcionalno_dinamično_uravnavanje_izražanja_genov_v_E._coli Reverzibilna toplotna regulacija za bifunkcionalno dinamično uravnavanje izražanja genov v E. coli] (Urška Fajdiga) &lt;br&gt;<br /> 9[http://wiki.fkkt.unilj.si/index.php/Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena%2C_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih] (Paula Horvat) &lt;br&gt;<br /> <br /> NAGRAJENI ŠTUDENTSKI PROJEKTI <br /> <br /> (Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do wiki strani študentske ekipe, katere projekt opisujete.) &lt;br&gt;<br /> <br /> 1 [http://wiki.fkkt.uni-lj.si/index.php/RESHAPE_-_spreminjanje_morfologije_nitastih_gliv RESHAPE - spreminjanje morfologije nitastih gliv] <br /> (Špela Supej) &lt;br&gt;<br /> 2 [http://wiki.fkkt.uni-lj.si/index.php/The_Chlamy_Cleaner:_razgradnja_pesticida_z_zeleno_algo The Chlamy Cleaner: razgradnja pesticida z zeleno algo] (Doroteja Armič) &lt;br&gt;<br /> 3 [http://wiki.fkkt.uni-lj.si/index.php/TheraPUFA:_nazalni_probiotik_proti_okužbam_in_vnetjem TheraPUFA- nazalni probiotik proti okužbam in vnetjem] (Barbara Slapnik) &lt;br&gt;<br /> 4 [[S-POP: Modularni biosenzor za zaznavanje obstojnih organskih onesnaževal v okoljskih vodah]] (Tadej Medved) &lt;br&gt;<br /> 5 [http://wiki.fkkt.uni-lj.si/index.php/MARS-magnetni_sistem_za_recikliranje_ATP MARS-magnetni sistem za recikliranje ATP] (David Miškić) &lt;br&gt;<br /> 6 [[B.O.T.: Bakterijska oscilacijska terapija za zdravljenje kolorektalnega raka]] (Neža Pavko) &lt;br&gt;<br /> 7 [[iGEMINI: Kvasovke kot prehransko dopolnilo v vesolju]] (Klementina Polanec) &lt;br&gt;<br /> 8 [[Antea-Glyphosate: Detekcija in razgradnja glifosata]] (Jernej Imperl) &lt;br&gt;<br /> ----<br /> <br /> Povzetki v slovenščini naj imajo 1200-1500 besed (viri v to vsoto ne štejejo). Povzetek je treba objaviti dva dni pred predstavitvijo do polnoči (za seminarje v sredo torej v ponedeljek). Predstavitev seminarja naj bo dolga 15 minut (13-17). Sledila bo razprava, ki praviloma ne bo daljša od 5 minut.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Sinteti%C4%8Dna_optogenetska_naprava_na_osnovi_BRET_za_pulzirajo%C4%8Do_ekspresijo_transgena,_ki_omogo%C4%8Da_glukozno_homeostazo_pri_mi%C5%A1ih&diff=18555 2021-04-19T21:53:10Z

<p>Paulahorvat: New page: == INTRODUCTION == Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transc...</p> <hr /> <div>== INTRODUCTION ==<br /> <br /> Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transcription factors and many other regulatory proteins are regulated in a pulsatile fashion, leading to a variety of phenotypic consequences, ranging from stress response to differentiation and signaling. Pulsing has been observed in many types of proteins. In alternative bacterial sigma factors, but also in mammalian tumor suppressors like p53. Mammalian cells exhibit many pulsatile factors. The stress response pathways mediated by p53, which controls the DNA damage response pulses, just as nuclear factor κB which is involved in immune responses. Iti s still unknown what functional capabilities can this pulsatile dynamic regulation provide for cells. Chemical inducers were used to dynamically regulate transgene expression. Slow degradation and metabolisation of chemical inducers has been shown as not so good application for controlling the dynamics of regulators in most pulsing systems. Therefore, more precise light-inducible systems have been developed that allow control of gene expression. Pulsatile dynamic regulation of regulators is achieved. The limitation that occurs is supplying adequate light intensity to activate these optogenetic systems. To solve this problem luminiscent opsins were developed ( directly coupling bioluminescent proteins to conventional light-sensitive opsins). BRET or bioluminescence resonance energy transfer is how luciferase substrates create bioluminiscence and activate opsins. After some time inactivation of opsins occurs and they regulate the cell activity in a pulsatile fashion. That is where the idea for transgene expression system that allows pulsatile regulation of gene expression both in vitro and in vivo came from. <br /> <br /> == EXPERIMENT ==<br /> <br /> In this experiment the development of the luminGAVPO, a luminsecent transcription factor is explained. LuminGAVPO enables pulsatile and quantitative activation of transgene expression in both mammalian cells and animals in a furimazine- or light irradiance-dependent manner. By controlling blood glucose homeostasis in type 1 diabetic mice its usefulness is tested. <br /> <br /> == RESULTS ==<br /> <br /> Design and construction of the LuminON transgene expression system.<br /> NanoLuc (Nluc) is a engineered luciferase from a deep sea shrimp, which is stable, bright, small and with sustained luminescence. It is used as luminiscence donor ( produces blue light) to activate LOV-based (The light-oxygen-voltage proteins) optogenetic modules. Nluc was fused to the N terminus of the mCherry-LOV2 fusion protein. When a protein was coexpressed with a mitochondria-anchored mVenus-Zdk protein, both mCherry and mVenus fluorescence accumulated at mitochondria under dark conditions. But the mCherry fluorescence readily diffused to the cytosol within 5 seconds after the addition of furimazine or exposure to external blue ligh. Blue light-inducible transgene expression systems based on Vivid were developed in which transcription factors bind to the promoter region ( to activate or repress gene transcription upon blue light exposure). The next step was to create a luminiscent transcription factor of a chimeric protein that consists of Nluc and a light-switchable transcription factor, GAVPO. Nluc-GAVPO fusions activated Gluc expression upon incubation with furimazine (FZ) or illumination with external blue light. On the other hand expression of Nluc and GAVPO saparately yielded only a response to external blue light. Further studies showed also that Nluc-GAVPO fusions driven by a strong CMV promoter had higher activation levels than those driven by a weak SV40 promoter. . Since Nluc-GAVPO with NG configuration had moderate activation levels yet exhibited maximal induction ratio by furimazine, we termed it luminGAVPO (luminescent GAVPO), and the transgene expression system based on luminGAVPO was named the LuminON system.Based on the activation information “OR”-like logic gate is predicted for luminGAVPO-activated transgene expression using furimazine and external blue light as the two inputs. It was also noticed that higher concentrations of furimazine could produce brighter and more sustained luminescence, but Gluc expression slightly decreased when the furimazine concentration is higher than 5 μM. In addition, highly precise control of transgene expression was achieved by combining the furimazine concentration and light irradiance. Another assumption was made, that induction by furimazine multiple times could increase the luminGAVPOmediated activation levels, considering that Nluc is resistant to autoinhibition by its catalytic byproducts. The results showed that Gluc expression increased with increasing round of induction by furimazine.<br /> <br /> Pulsatile activation of transgene expression mediated by luminGAVPO.<br /> Furimazine-induced transgene expression by luminGAVPO would display a pulsatile profile ( activation followed by inactivation). After furimazine induction, high levels of Gluc mRNA and protein production were observed. The results showed that the Gluc synthesis reached a maximum m at ~4–6 h after furimazine induction and then sharply decreased. The medium has also been changed to remove the residual furimazine at 4 h after induction. Therefore, it can be seen that the majority of the furimazine had been consumed within 4 h after induction. It can be concluded that the pulse amplitude and duration of transgene expression were highly dependent on the dose of furimazine. Lower doses of furimazine exhibited shorter durations and smaller amplitudes of pulsatile activation. <br /> <br /> Pulsatile activation of transgene expression in mice.<br /> These results in vitro needed to be proved in vivo. Therefore, luminGAVPO and mCherry reporter plasmids were transfected into the livers of mice. This has been done using a hydrodynamic procedure, which is a rapid injection of a large volume of solution into a vasculature to facilitate substance transfer into parenchyma cells. Mice were administrated furimazine via tail intravenous (IV), intraperitoneal (IP) or intragastric. mCherry fluorescence was observed in the livers of these mice. After the measured time, the results showed that Gluc expression reached a maximum level within 4 h after furimazine induction and decreased after that. It can be concluded that the half-life of Gluc in the bloodstream is shorter than in the culture medium, and a lower dose of furimazine exhibited shorter durations and smaller amplitudes of Gluc expression in the bloodstream. After all these results demonstrate that the LuminON system can be used for pulsatile and reversible activation of transgene expression in vivo.<br /> <br /> Pulsatile expression of insulin to enhance blood glucose homeostasis in T1D mice.<br /> Insulin is a hormon synthesized by the β-cells of pancreas and is a regulator of metabolism because it maintains glucose homeostasis. This pulsatile secretion of insulin may match the expression profile mediated by luminGAVPO. HEKFUR-Gluc-P2A-mINS cells were engineered for luminGAVPO-mediated insulin production. luminGAVPO and UASG-TATA-Gluc-P2A-mINS expression cassettes were integrated into HEK293T cells using the Sleeping Beauty transposon system, which is a promising technology platform for gene transfer in vertebrates. After that clones with the highest Gluc expression were microencapsulated into coherent, semipermeable and immunoprotective alginate-poly-(L-lysine)-alginate beads that allow free diffusion of substances with low molecular weight. T1D mice receiving the microencapsulated cells were treated with 5 mg/kg furimazine, and their blood glucose levels were measured after 3 h. The results showed that , a higher dose of furimazine resulted in more significant restoration of blood glucose and prolonged the maintenance of glucose homeostasis. The results showed the potential in the oral administration of furimazine instead of traditional intramuscular injection of insulin before and after meals.<br /> <br /> == DISCUSSION ==<br /> <br /> Nluc is an ideal luminescence donor to activate LOV protein-based optogenetic systems via BRET, because it has the advantages of high brightness, no requirement of ATP and resistance to autoinhibition by its catalytic byproducts and perfectly matches the absorption spectra of the light-sensitive LOV proteins. In this study BRET-based transgene expression system was used fusing Nluc to a light-switchable transcription factor. Induction by furimazine caused activation of the proximal VVD domain in luminGAVPO, resulting in dimerization of luminGAVPO and binding of luminGAVPO to its cognate promoter, thereby initiating transcription of the target gene. The luminGAVPO dimer gradually dissociates from the promoter, leading to inactivation of transcription, after the furimazine is consumed. That is why this transgene expression is called pulsatile. LuminON system was also used to control the pulsatile and repetitive expression of insulin to enhance blood-glucose homeostasis in T1D mice, which could prevent the hypoglycemia. To conclude, BRET-based optogenetic device developed in this study can be used to precisely control the dynamics of key signaling proteins in a pulsatile fashion both in vitro and in vivo.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Razvoj_DNA_cepiva_proti_metastazam_melanoma_z_vdihavanjem_na_podlagi_analize_zna%C4%8Dilnosti_transgene_ekspresije_gole_pDNA_in_trojni_kompleks_v_mi%C5%A1jem_plju%C4%8Dnem_tkivu&diff=18023 2021-03-22T23:33:34Z

<p>Paulahorvat: /* Literature */</p> <hr /> <div>==Melanoma==<br /> <br /> Melanoma is the deadliest form of skin cancer. After reaching metastasis, survival rates drop, therefore early diagnosis and a certain lifestyle greatly reduce the risk.[1] Potential causes for melanoma are: family history, personal characteristic ( pale skin or bright eye color ), sun exsposure and atypical (asymmetric) mole.[2]<br /> <br /> ==DNA vaccines==<br /> <br /> Protein antigens are encoded into DNA, during DNA vaccine production. Antigen-specific humoral and cell-mediated immunity are induced. A great number of melanoma antignes have been repoted, which is why DNA vaccines have potential in prevention and treatment of melanoma. DNA vaccines are produced because they are versatile, inexpensive, easy to manufacture, easy to store and safe to handle. Pulmonary administration route (inhalation) is used in this experiment, because the lungs contain highly responsive immune system.[3]<br /> <br /> ==Materials and methods==<br /> <br /> Model pDNA (pCMV-Luc, pEGFP-C1, or pZsGreen) was used to evaluate transfection efficacy after inhalation in mice. Gene expression of naked pDNA and gene expression of the ternary complexes were compared.<br /> 1) Pulmonary administration of naked pCMV-Luc or the ternary complex containing the pCMV-Luc was performed to monitore gene expression. 6, 24 and 48 hours after administration liver, kidneys, spleen, heart and lungs were observed. Luciferase activity was measured and was indicated as relative light units (RLUs) per gram of tissue.<br /> 2)Naked pEGFP-C1 and ternary complexes containing pEGFP-C1 were administrated intrapulmonary to visualize the accumulation and gene expression of the complexes. 24 hours after the administration GFP expression levels in the lungs were observed using fluorescent microscopy.<br /> 3) Naked pZsGreen1-N1 and the ternary complex containing pZsGreen1-N1 were used too as a model pDNA. In addition DiD and Dil in 5% glusoce were administrated too. This step was done by multicolor deep imaging system using the tissue clearing reagent.[3]<br /> <br /> Naked pDNA, ternary complexes containing pUb-M or ternanry complexes containing pEGFP-C1 were administrated three times biweekly. Luciferase activity and tumor volume was monitored, becuse it was belived that it will prevent melanoma metastasis.<br /> Th1 Cytokine (IFN-gama, TNF-alpha, and IL-6 ) secretion was monitored too.[3]<br /> <br /> ==Results==<br /> <br /> Luciferase activity in the lungs 6 and 24 h after administration of naked pCMV-Luc and the ternary complex was significantly higher than that in other organs.<br /> After the administration with naked pEGFP-C1 weak fluorescence was noticed. On the other hand, after the administration with the ternary complex containing pEGFP-C1 strong fluorescence was noticed.<br /> ZsGreen1 expression was noticed in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in the lungs.<br /> All three, pEGFP-C1 complex, naked pUb-M and the Pub-M complex inhibited the lung metastasis, according to reduced luciferase activity and reduced tumor growth. The most effective of all was the pUb-M complex, and the least effective was pEGFP-C1 complex.<br /> Splenic cells immunized by the Pub-M complex secreted the largest amounts of IFN-gama, TNF-alpha, and IL-6.[3]<br /> <br /> ==Conclusions==<br /> <br /> Pulmonary administration is shown to be an effective route for the delivery of DNA vaccines. <br /> Both, naked pDNA and the ternary complex of pDNA showed high gene expression in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in lungs. Since the ternary complex showed stronger effect, it can be concluded that it can be efficiently taken up by alveolar macrophages than naked pDNA. <br /> The pUb-M complex is the most effective in the melanoma metastasis inhibition, as same as in the secretion of Th1 cytokines (IFN-gama and TNF-alpha).[3]<br /> <br /> ==Literature==<br /> <br /> 1.Davis LE, Shalin SC, Tackett AJ. Current state of melanoma diagnosis and treatment. Cancer Biol Ther. 2019;20(11):1366-1379. doi:10.1080/15384047.2019.1640032<br /> <br /> 2.Heistein JB, Acharya U. Malignant Melanoma. [Updated 2020 Dec 5]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-.<br /> <br /> 3.Kodama, Yukinobu and Nakashima, Mikiro and Nagahara, Tadayuki and Oyama et. al, Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues, Pharmaceutics, 12, 2020.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=MBT_seminarji_2021&diff=18022 2021-03-22T23:32:08Z

<p>Paulahorvat: </p> <hr /> <div>Seminarji iz Molekularne biotehnologije so letos organizirani tako, da vsak študent (praviloma v paru, lahko pa tudi samostojno) obdela temo s področja cepiv proti virusu SARS-CoV-2 in o tem pripravi kratek poljudno napisan povzetek. Ta del seminarjev je predstavljen na [[protikovidna cepiva|ločeni strani]].<br /> V drugem delu vsak študent predstavi nek raziskovalni dosežek s širšega področja molekularne biotehnologije. Seznam tem in predstavitev za študijsko leto 2020/21 je predstavljen tu.<br /> <br /> Povzetke morate objaviti do torka do polnoči v tednu, ko imate seminar (v četrtek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. predlani).<br /> <br /> Način vnosa:<br /> <br /> The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops &amp; Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak (28.2.)&lt;br&gt;<br /> (slovenski naslov povežite z novo stranjo, na kateri bo povzetek)<br /> <br /> ----<br /> <br /> <br /> Naslovi odobrenih člankov po temah:<br /> <br /> '''Farmacevtsko pomembni proteini'''&lt;br&gt;<br /> # Development of Antibody-Fragment-Producing Rice for Neutralization of Human Norovirus (A. Sasou ''et. al''; Frontiers in Plant Science 12, 2021; https://doi.org/10.3389/fpls.2021.639953). [[Proizvodnja riža za sintezo fragmentov protiteles proti humanemu norovirusu.]] Mateja Žvipelj (11.3.)<br /> # A New Plant Expression System for Producing Pharmaceutical Proteins (N. Abd-Aziz ''et. al''; Molecular Biotechnology 62, 2020; https://doi.org/10.1007/s12033-020-00242-2). [[Razvoj ekspresijskega sistema za proizvodnjo farmacevtskih proteinov v rastlini Mucuna bracteata]]. Jernej Imperl (18.3.)<br /> # Development of a Recombinant Monospecific Anti-PLGF Bivalent Nanobody and Evaluation of it in Angiogenesis Modulation (A. Nikooharf &quot;et all&quot;; Molecular Biotechnology 62, 2020; https://link.springer.com/article/10.1007/s12033-020-00275-7#additional-information) [[Razvoj rekombinantnih monospecifičnih bivalentnih nanoteles proti PLGF-u]]. Nika Zaveršek (18.3.)<br /> <br /> '''Cepiva'''&lt;br&gt;<br /> # Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues (Kodama ''et.al'';Pharmaceutics 12,2020; https://www.mdpi.com/1999-4923/12/6/540#framed_div_cited_count) [[ Razvoj DNA cepiva proti metastazam melanoma z vdihavanjem na podlagi analize značilnosti transgene ekspresije gole pDNA in trojni kompleks v mišjem pljučnem tkivu]]. Paula Horvat (25.3.)&lt;br&gt;<br /> # Neža Pavko (25.3.)&lt;br&gt;<br /> <br /> '''Gensko spremenjene rastline'''&lt;br&gt;<br /> # (1.4.)&lt;br&gt;<br /> # (1.4.)&lt;br&gt;<br /> # Andrej Race (7.4.)<br /> # Peter Škrinjar (7.4.)<br /> <br /> '''Gensko spremenjene živali in celične linije'''&lt;br&gt;<br /> # Urša Lovše (8.4.)<br /> # Matija Ruparčič (8.4.)<br /> <br /> '''Nizkomolekularni biotehnološki produkti'''&lt;br&gt;<br /> # Saša Slabe (14.4.)<br /> # Luka Gnidovec (15.4.)<br /> # Liza Ulčakar (15.4.)<br /> <br /> '''Biotehnološki polimeri'''&lt;br&gt;<br /> # Anže Karlek (21.4.)<br /> # Ana Maklin (22.4.)<br /> # Urban Hribar(22.4.)<br /> <br /> '''Biotehnološko pridobljeni encimi'''&lt;br&gt;<br /> # Urška Fajdiga (5.5.)<br /> # Mirsad Mešić (6.5.)<br /> # Martina Lokar (6.5.)<br /> <br /> '''Metabolno inženirstvo v biotehnologiji'''&lt;br&gt;<br /> # Jerneja Nimac (12.5.)<br /> # Urška Pečarič Strnad (12.5.)<br /> # Klementina Polanec (13.5.)<br /> # Ernestina Lavrih (13.5.)<br /> <br /> '''Biomasa in biogoriva'''&lt;br&gt;<br /> # Željka Erić (19.5.)<br /> # Karin Dobravc Škof (20.5.)<br /> # Katja Doberšek(20.5.)<br /> <br /> '''Okoljski vidiki biotehnologije in bioremediacija'''&lt;br&gt;<br /> # Almina Tahirović (26.5.)<br /> # Eva Keber (27.5.)<br /> # Nina Lukančič (27.5.)</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Razvoj_DNA_cepiva_proti_metastazam_melanoma_z_vdihavanjem_na_podlagi_analize_zna%C4%8Dilnosti_transgene_ekspresije_gole_pDNA_in_trojni_kompleks_v_mi%C5%A1jem_plju%C4%8Dnem_tkivu&diff=18021 2021-03-22T23:23:55Z

<p>Paulahorvat: /* Literature */</p> <hr /> <div>==Melanoma==<br /> <br /> Melanoma is the deadliest form of skin cancer. After reaching metastasis, survival rates drop, therefore early diagnosis and a certain lifestyle greatly reduce the risk.[1] Potential causes for melanoma are: family history, personal characteristic ( pale skin or bright eye color ), sun exsposure and atypical (asymmetric) mole.[2]<br /> <br /> ==DNA vaccines==<br /> <br /> Protein antigens are encoded into DNA, during DNA vaccine production. Antigen-specific humoral and cell-mediated immunity are induced. A great number of melanoma antignes have been repoted, which is why DNA vaccines have potential in prevention and treatment of melanoma. DNA vaccines are produced because they are versatile, inexpensive, easy to manufacture, easy to store and safe to handle. Pulmonary administration route (inhalation) is used in this experiment, because the lungs contain highly responsive immune system.[3]<br /> <br /> ==Materials and methods==<br /> <br /> Model pDNA (pCMV-Luc, pEGFP-C1, or pZsGreen) was used to evaluate transfection efficacy after inhalation in mice. Gene expression of naked pDNA and gene expression of the ternary complexes were compared.<br /> 1) Pulmonary administration of naked pCMV-Luc or the ternary complex containing the pCMV-Luc was performed to monitore gene expression. 6, 24 and 48 hours after administration liver, kidneys, spleen, heart and lungs were observed. Luciferase activity was measured and was indicated as relative light units (RLUs) per gram of tissue.<br /> 2)Naked pEGFP-C1 and ternary complexes containing pEGFP-C1 were administrated intrapulmonary to visualize the accumulation and gene expression of the complexes. 24 hours after the administration GFP expression levels in the lungs were observed using fluorescent microscopy.<br /> 3) Naked pZsGreen1-N1 and the ternary complex containing pZsGreen1-N1 were used too as a model pDNA. In addition DiD and Dil in 5% glusoce were administrated too. This step was done by multicolor deep imaging system using the tissue clearing reagent.[3]<br /> <br /> Naked pDNA, ternary complexes containing pUb-M or ternanry complexes containing pEGFP-C1 were administrated three times biweekly. Luciferase activity and tumor volume was monitored, becuse it was belived that it will prevent melanoma metastasis.<br /> Th1 Cytokine (IFN-gama, TNF-alpha, and IL-6 ) secretion was monitored too.[3]<br /> <br /> ==Results==<br /> <br /> Luciferase activity in the lungs 6 and 24 h after administration of naked pCMV-Luc and the ternary complex was significantly higher than that in other organs.<br /> After the administration with naked pEGFP-C1 weak fluorescence was noticed. On the other hand, after the administration with the ternary complex containing pEGFP-C1 strong fluorescence was noticed.<br /> ZsGreen1 expression was noticed in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in the lungs.<br /> All three, pEGFP-C1 complex, naked pUb-M and the Pub-M complex inhibited the lung metastasis, according to reduced luciferase activity and reduced tumor growth. The most effective of all was the pUb-M complex, and the least effective was pEGFP-C1 complex.<br /> Splenic cells immunized by the Pub-M complex secreted the largest amounts of IFN-gama, TNF-alpha, and IL-6.[3]<br /> <br /> ==Conclusions==<br /> <br /> Pulmonary administration is shown to be an effective route for the delivery of DNA vaccines. <br /> Both, naked pDNA and the ternary complex of pDNA showed high gene expression in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in lungs. Since the ternary complex showed stronger effect, it can be concluded that it can be efficiently taken up by alveolar macrophages than naked pDNA. <br /> The pUb-M complex is the most effective in the melanoma metastasis inhibition, as same as in the secretion of Th1 cytokines (IFN-gama and TNF-alpha).[3]<br /> <br /> ==Literature==<br /> <br /> 1.Davis LE, Shalin SC, Tackett AJ. Current state of melanoma diagnosis and treatment. Cancer Biol Ther. 2019;20(11):1366-1379. doi:10.1080/15384047.2019.1640032<br /> <br /> 2.Heistein JB, Acharya U. Malignant Melanoma. [Updated 2020 Dec 5]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-.<br /> <br /> 3.Kodama, Yukinobu and Nakashima, Mikiro and Nagahara, Tadayuki and Oyama et al., Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues, Pharmaceutics, 12, 2020.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=Razvoj_DNA_cepiva_proti_metastazam_melanoma_z_vdihavanjem_na_podlagi_analize_zna%C4%8Dilnosti_transgene_ekspresije_gole_pDNA_in_trojni_kompleks_v_mi%C5%A1jem_plju%C4%8Dnem_tkivu&diff=18020 2021-03-22T23:23:23Z

<p>Paulahorvat: New page: ==Melanoma== Melanoma is the deadliest form of skin cancer. After reaching metastasis, survival rates drop, therefore early diagnosis and a certain lifestyle greatly reduce the risk.[1] P...</p> <hr /> <div>==Melanoma==<br /> <br /> Melanoma is the deadliest form of skin cancer. After reaching metastasis, survival rates drop, therefore early diagnosis and a certain lifestyle greatly reduce the risk.[1] Potential causes for melanoma are: family history, personal characteristic ( pale skin or bright eye color ), sun exsposure and atypical (asymmetric) mole.[2]<br /> <br /> ==DNA vaccines==<br /> <br /> Protein antigens are encoded into DNA, during DNA vaccine production. Antigen-specific humoral and cell-mediated immunity are induced. A great number of melanoma antignes have been repoted, which is why DNA vaccines have potential in prevention and treatment of melanoma. DNA vaccines are produced because they are versatile, inexpensive, easy to manufacture, easy to store and safe to handle. Pulmonary administration route (inhalation) is used in this experiment, because the lungs contain highly responsive immune system.[3]<br /> <br /> ==Materials and methods==<br /> <br /> Model pDNA (pCMV-Luc, pEGFP-C1, or pZsGreen) was used to evaluate transfection efficacy after inhalation in mice. Gene expression of naked pDNA and gene expression of the ternary complexes were compared.<br /> 1) Pulmonary administration of naked pCMV-Luc or the ternary complex containing the pCMV-Luc was performed to monitore gene expression. 6, 24 and 48 hours after administration liver, kidneys, spleen, heart and lungs were observed. Luciferase activity was measured and was indicated as relative light units (RLUs) per gram of tissue.<br /> 2)Naked pEGFP-C1 and ternary complexes containing pEGFP-C1 were administrated intrapulmonary to visualize the accumulation and gene expression of the complexes. 24 hours after the administration GFP expression levels in the lungs were observed using fluorescent microscopy.<br /> 3) Naked pZsGreen1-N1 and the ternary complex containing pZsGreen1-N1 were used too as a model pDNA. In addition DiD and Dil in 5% glusoce were administrated too. This step was done by multicolor deep imaging system using the tissue clearing reagent.[3]<br /> <br /> Naked pDNA, ternary complexes containing pUb-M or ternanry complexes containing pEGFP-C1 were administrated three times biweekly. Luciferase activity and tumor volume was monitored, becuse it was belived that it will prevent melanoma metastasis.<br /> Th1 Cytokine (IFN-gama, TNF-alpha, and IL-6 ) secretion was monitored too.[3]<br /> <br /> ==Results==<br /> <br /> Luciferase activity in the lungs 6 and 24 h after administration of naked pCMV-Luc and the ternary complex was significantly higher than that in other organs.<br /> After the administration with naked pEGFP-C1 weak fluorescence was noticed. On the other hand, after the administration with the ternary complex containing pEGFP-C1 strong fluorescence was noticed.<br /> ZsGreen1 expression was noticed in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in the lungs.<br /> All three, pEGFP-C1 complex, naked pUb-M and the Pub-M complex inhibited the lung metastasis, according to reduced luciferase activity and reduced tumor growth. The most effective of all was the pUb-M complex, and the least effective was pEGFP-C1 complex.<br /> Splenic cells immunized by the Pub-M complex secreted the largest amounts of IFN-gama, TNF-alpha, and IL-6.[3]<br /> <br /> ==Conclusions==<br /> <br /> Pulmonary administration is shown to be an effective route for the delivery of DNA vaccines. <br /> Both, naked pDNA and the ternary complex of pDNA showed high gene expression in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in lungs. Since the ternary complex showed stronger effect, it can be concluded that it can be efficiently taken up by alveolar macrophages than naked pDNA. <br /> The pUb-M complex is the most effective in the melanoma metastasis inhibition, as same as in the secretion of Th1 cytokines (IFN-gama and TNF-alpha).[3]<br /> <br /> ==Literature==<br /> <br /> 1.Davis LE, Shalin SC, Tackett AJ. Current state of melanoma diagnosis and treatment. Cancer Biol Ther. 2019;20(11):1366-1379. doi:10.1080/15384047.2019.1640032<br /> 2.Heistein JB, Acharya U. Malignant Melanoma. [Updated 2020 Dec 5]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-.<br /> 3.Kodama, Yukinobu and Nakashima, Mikiro and Nagahara, Tadayuki and Oyama et al., Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues, Pharmaceutics, 12, 2020.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=BNT-seminar&diff=17932 2021-03-11T23:42:51Z

<p>Paulahorvat: </p> <hr /> <div>= Bionanotehnologija 2021- seminar =<br /> doc. dr. Gregor Gunčar, K2.022<br /> <br /> == Seznam seminarjev ==<br /> <br /> {| {{table}}<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''Vpisna številka'''<br /> 30170005 &lt;br /&gt;<br /> 30019058 &lt;br /&gt;<br /> 30200303 &lt;br /&gt;<br /> 30019363 &lt;br /&gt;<br /> 30019057 &lt;br /&gt;<br /> 30170131 &lt;br /&gt;<br /> 30170078 &lt;br /&gt;<br /> 30019040 &lt;br /&gt;<br /> 30170177 &lt;br /&gt;<br /> 30200324 &lt;br /&gt;<br /> 30019063 &lt;br /&gt;<br /> 30170103 &lt;br /&gt;<br /> 30170002 &lt;br /&gt;<br /> 30200319 &lt;br /&gt;<br /> 30200309 &lt;br /&gt;<br /> 30200320 &lt;br /&gt;<br /> 30019056 &lt;br /&gt;<br /> 30200311 &lt;br /&gt;<br /> 30200306 &lt;br /&gt;<br /> 30170243 &lt;br /&gt;<br /> 30019051 &lt;br /&gt;<br /> 30170141 &lt;br /&gt;<br /> 30170061 &lt;br /&gt;<br /> 30019035 &lt;br /&gt;<br /> 30200316 &lt;br /&gt;<br /> 30170222 &lt;br /&gt;<br /> 30200317 &lt;br /&gt;<br /> 30170193 &lt;br /&gt;<br /> 30200315 &lt;br /&gt;<br /> 30200307 &lt;br /&gt;<br /> 30200321 &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''datum predstavitve'''<br /> 16.3.2021 &lt;br /&gt;<br /> 16.3.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 30.3.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 20.4.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 20.4.2020 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> 30.3.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''recenzent1'''<br /> Anamarija Agnič &lt;br /&gt;<br /> Aljaž Bratina &lt;br /&gt;<br /> Urban Hribar &lt;br /&gt;<br /> Sabina Sladič Oblak &lt;br /&gt;<br /> Barbara Slapnik &lt;br /&gt;<br /> Tina Kolenc Milavec &lt;br /&gt;<br /> Klementina Polanec &lt;br /&gt;<br /> Ajda Godec &lt;br /&gt;<br /> Martin Špendl &lt;br /&gt;<br /> Tjaša Mlakar &lt;br /&gt;<br /> Sara Laznik &lt;br /&gt;<br /> Martina Lokar &lt;br /&gt;<br /> Doroteja Armič &lt;br /&gt;<br /> Martin Špendl &lt;br /&gt;<br /> Saša Slabe &lt;br /&gt;<br /> Mateja Žvipelj &lt;br /&gt;<br /> Špela Supej &lt;br /&gt;<br /> Urška Fajdiga &lt;br /&gt;<br /> Ernestina Lavrih &lt;br /&gt;<br /> Nika Mikulič Vernik &lt;br /&gt;<br /> Liza Ulčakar &lt;br /&gt;<br /> Urša Štrancar &lt;br /&gt;<br /> Tadej Medved &lt;br /&gt;<br /> Urška Zagorc &lt;br /&gt;<br /> Nina Lukančič &lt;br /&gt;<br /> Urška Pečarič Strnad &lt;br /&gt;<br /> Anže Karlek &lt;br /&gt;<br /> Luka Gnidovec &lt;br /&gt;<br /> Urša Lovše &lt;br /&gt;<br /> Almina Tahirović &lt;br /&gt;<br /> Mirsad Mešić &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''recenzent2'''<br /> Urška Pečarič Strnad &lt;br /&gt;<br /> Urška Zagorc &lt;br /&gt;<br /> Liza Ulčakar &lt;br /&gt;<br /> Urša Štrancar &lt;br /&gt;<br /> Sara Laznik &lt;br /&gt;<br /> Luka Gnidovec &lt;br /&gt;<br /> Irma Zeljković &lt;br /&gt;<br /> Eva Keber &lt;br /&gt;<br /> Jernej Imperl &lt;br /&gt;<br /> Barbara Slapnik &lt;br /&gt;<br /> Urša Lovše &lt;br /&gt;<br /> Mateja Žvipelj &lt;br /&gt;<br /> Neža Pavko &lt;br /&gt;<br /> Almina Tahirović &lt;br /&gt;<br /> Nika Mikulič Vernik &lt;br /&gt;<br /> Urška Fajdiga &lt;br /&gt;<br /> Saša Slabe &lt;br /&gt;<br /> Klementina Polanec &lt;br /&gt;<br /> Tjaša Mlakar &lt;br /&gt;<br /> Ajda Godec &lt;br /&gt;<br /> Jernej Imperl &lt;br /&gt;<br /> Špela Supej &lt;br /&gt;<br /> Aljaž Bratina &lt;br /&gt;<br /> Nina Lukančič &lt;br /&gt;<br /> Matija Ruparčič &lt;br /&gt;<br /> Anže Karlek &lt;br /&gt;<br /> Sabina S. Oblak &lt;br /&gt;<br /> Urban Hribar &lt;br /&gt;<br /> Mirsad Mešić &lt;br /&gt;<br /> Tina Kolenc Milavec &lt;br /&gt;<br /> Tadej Medved &lt;br /&gt;<br /> |}<br /> <br /> ==Naloga==<br /> Pripravite projektno nalogo iz področja Bionanotehnologije. Najpomembnejša je originalna ideja za nek izvedljiv projekt, ki pa mora biti takšen, da pritegne investitorje. Ker je pomembno tudi kako boste to naredili, morate predstaviti tudi metodo in ne samo ideje. Natančno morate vedeti, kako boste projekt izvedli.<br /> <br /> Predlagana struktura teksta:<br /> * Uvod<br /> * Predstavitev problema, znanstvena izhodišča, cilji<br /> * Izvedba projekta, metodologija, tehnike, materiali, vprašanja, hipoteze<br /> * Literatura<br /> <br /> Za pripravo seminarja velja naslednje:&lt;br&gt;<br /> * Elektronska verzija seminarja: avtor, naslov projekta, razširjeni povzetek projekta- 350-400 besed (brez literature) in grafični povzetek (čez približno pol strani). Vse naj bo na maksimalno dveh straneh, a ne sme vsebovati manj kot 350 besed (sem se ne šteje literatura). <br /> * Elektronsko verzijo seminarja oddajte en dan pred predstavitvijo, kasneje pa boste vsebino še prekopirali na za to določeno spletno stran, predstavitev pa eno uro pred seminarjem na [http://bio.ijs.si/~zajec/poslji/ strežnik].<br /> * Vsi seminarji so v elektronski obliki dostopni [http://bio.ijs.si/~zajec/poslji/bioseminar/ tukaj].<br /> * Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo XY minut. Recenzenti morajo biti na predstavitvi prisotni.<br /> * Predstavitvi sledi razprava. Recenzenta morate predlagati vsaj eno izboljšavo predstavljenega projekta.<br /> <br /> ==&lt;font color=green&gt;Imena datotek&lt;/font&gt;==<br /> Prosim vas, da vse datoteke poimenujete po naslednjem modelu:<br /> * 20_nano_Priimek.doc za seminar, npr. 20_nano_Craik_Venter.doc<br /> * 20_nano_Priimek.ppt za prezentacijo, npr. 20_nano_Craik_Venter.ppt<br /> <br /> ==Urejanje spletnih strani na wikiju==<br /> Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''<br /> <br /> ==Citiranje virov==<br /> Citiranje je možno po več shemah, važno je, da se držite ene same. V seminarskih nalogah in diplomskih nalogah FKKT uprabljajte shemo citiranja, ki je pobarvana &lt;font color=green&gt;zeleno&lt;/font&gt;.<br /> Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.<br /> Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati.&lt;br&gt;<br /> '''Citiranje knjig:'''&lt;br&gt;<br /> Priimek, I. ''Naslov''. Kraj: Založba, letnica.&lt;br&gt;<br /> Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN.&lt;br&gt;<br /> <br /> Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005.&lt;br&gt;<br /> Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4.&lt;br&gt;<br /> Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.). <br /> <br /> '''Citiranje člankov:'''&lt;br&gt;<br /> Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani.&lt;br&gt;<br /> &lt;font color=green&gt;Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., Smith, H. O. in Venter, J. C.<br /> Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, 317, str. 632-638.&lt;/font&gt;<br /> <br /> Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole:&lt;br&gt;<br /> Priimek, I. (letnica, številka). Naslov. Naslov revije, strani.&lt;br&gt;<br /> Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.<br /> <br /> Revija Science uporablja skrajšani zapis:&lt;br&gt;<br /> C. Lartigue ''et al''. Science 317, 632 (2007)&lt;br&gt;<br /> <br /> V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne). Navesti morate tudi vse avtorje dela, razen v primeru, ko jih je 10 ali več. Takrat navedite le prvih devet, za ostale pa uporabite okrajšavo in sod. (in sodelavci). Pred zadnjim avtorjem naj bo vedno besedica &quot;in&quot;. <br /> <br /> <br /> '''Citiranje spletnih virov:'''&lt;br&gt;<br /> Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov&lt;br&gt;<br /> strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life&lt;br&gt;<br /> Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=BNT-seminar&diff=17931 2021-03-11T23:39:24Z

<p>Paulahorvat: </p> <hr /> <div>= Bionanotehnologija 2021- seminar =<br /> doc. dr. Gregor Gunčar, K2.022<br /> <br /> == Seznam seminarjev ==<br /> <br /> {| {{table}}<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''Vpisna številka'''<br /> 30170005 &lt;br /&gt;<br /> 30019058 &lt;br /&gt;<br /> 30200303 &lt;br /&gt;<br /> 30019363 &lt;br /&gt;<br /> 30019057 &lt;br /&gt;<br /> 30170131 &lt;br /&gt;<br /> 30170078 &lt;br /&gt;<br /> 30019040 &lt;br /&gt;<br /> 30170177 &lt;br /&gt;<br /> 30200324 &lt;br /&gt;<br /> 30019063 &lt;br /&gt;<br /> 30170103 &lt;br /&gt;<br /> 30170002 &lt;br /&gt;<br /> 30200319 &lt;br /&gt;<br /> 30200309 &lt;br /&gt;<br /> 30200320 &lt;br /&gt;<br /> 30019056 &lt;br /&gt;<br /> 30200311 &lt;br /&gt;<br /> 30200306 &lt;br /&gt;<br /> 30170243 &lt;br /&gt;<br /> 30019051 &lt;br /&gt;<br /> 30170141 &lt;br /&gt;<br /> 30170061 &lt;br /&gt;<br /> 30019035 &lt;br /&gt;<br /> 30200316 &lt;br /&gt;<br /> 30170222 &lt;br /&gt;<br /> 30200317 &lt;br /&gt;<br /> 30170193 &lt;br /&gt;<br /> 30200315 &lt;br /&gt;<br /> 30200307 &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''datum predstavitve'''<br /> 16.3.2021 &lt;br /&gt;<br /> 16.3.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 30.3.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 20.4.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 20.4.2020 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> 30.3.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''recenzent1'''<br /> Anamarija Agnič &lt;br /&gt;<br /> Aljaž Bratina &lt;br /&gt;<br /> Urban Hribar &lt;br /&gt;<br /> Sabina Sladič Oblak &lt;br /&gt;<br /> Barbara Slapnik &lt;br /&gt;<br /> Tina Kolenc Milavec &lt;br /&gt;<br /> Klementina Polanec &lt;br /&gt;<br /> Ajda Godec &lt;br /&gt;<br /> Martin Špendl &lt;br /&gt;<br /> Tjaša Mlakar &lt;br /&gt;<br /> Sara Laznik &lt;br /&gt;<br /> Martina Lokar &lt;br /&gt;<br /> Doroteja Armič &lt;br /&gt;<br /> Martin Špendl &lt;br /&gt;<br /> Saša Slabe &lt;br /&gt;<br /> Mateja Žvipelj &lt;br /&gt;<br /> Špela Supej &lt;br /&gt;<br /> Urška Fajdiga &lt;br /&gt;<br /> Ernestina Lavrih &lt;br /&gt;<br /> Nika Mikulič Vernik &lt;br /&gt;<br /> Liza Ulčakar &lt;br /&gt;<br /> Urša Štrancar &lt;br /&gt;<br /> Tadej Medved &lt;br /&gt;<br /> Urška Zagorc &lt;br /&gt;<br /> Nina Lukančič &lt;br /&gt;<br /> Urška Pečarič Strnad &lt;br /&gt;<br /> Anže Karlek &lt;br /&gt;<br /> Luka Gnidovec &lt;br /&gt;<br /> Urša Lovše &lt;br /&gt;<br /> Almina Tahirović &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''recenzent2'''<br /> Urška Pečarič Strnad &lt;br /&gt;<br /> Urška Zagorc &lt;br /&gt;<br /> Liza Ulčakar &lt;br /&gt;<br /> Urša Štrancar &lt;br /&gt;<br /> Sara Laznik &lt;br /&gt;<br /> Luka Gnidovec &lt;br /&gt;<br /> Irma Zeljković &lt;br /&gt;<br /> Eva Keber &lt;br /&gt;<br /> Jernej Imperl &lt;br /&gt;<br /> Barbara Slapnik &lt;br /&gt;<br /> Urša Lovše &lt;br /&gt;<br /> Mateja Žvipelj &lt;br /&gt;<br /> Neža Pavko &lt;br /&gt;<br /> Almina Tahirović &lt;br /&gt;<br /> Nika Mikulič Vernik &lt;br /&gt;<br /> Urška Fajdiga &lt;br /&gt;<br /> Saša Slabe &lt;br /&gt;<br /> Klementina Polanec &lt;br /&gt;<br /> Tjaša Mlakar &lt;br /&gt;<br /> Ajda Godec &lt;br /&gt;<br /> Jernej Imperl &lt;br /&gt;<br /> Špela Supej &lt;br /&gt;<br /> Aljaž Bratina &lt;br /&gt;<br /> Nina Lukančič &lt;br /&gt;<br /> Matija Ruparčič &lt;br /&gt;<br /> Anže Karlek &lt;br /&gt;<br /> Sabina S. Oblak &lt;br /&gt;<br /> Urban Hribar &lt;br /&gt;<br /> Mirsad Mešić &lt;br /&gt;<br /> Tina Kolenc Milavec &lt;br /&gt;<br /> |}<br /> <br /> ==Naloga==<br /> Pripravite projektno nalogo iz področja Bionanotehnologije. Najpomembnejša je originalna ideja za nek izvedljiv projekt, ki pa mora biti takšen, da pritegne investitorje. Ker je pomembno tudi kako boste to naredili, morate predstaviti tudi metodo in ne samo ideje. Natančno morate vedeti, kako boste projekt izvedli.<br /> <br /> Predlagana struktura teksta:<br /> * Uvod<br /> * Predstavitev problema, znanstvena izhodišča, cilji<br /> * Izvedba projekta, metodologija, tehnike, materiali, vprašanja, hipoteze<br /> * Literatura<br /> <br /> Za pripravo seminarja velja naslednje:&lt;br&gt;<br /> * Elektronska verzija seminarja: avtor, naslov projekta, razširjeni povzetek projekta- 350-400 besed (brez literature) in grafični povzetek (čez približno pol strani). Vse naj bo na maksimalno dveh straneh, a ne sme vsebovati manj kot 350 besed (sem se ne šteje literatura). <br /> * Elektronsko verzijo seminarja oddajte en dan pred predstavitvijo, kasneje pa boste vsebino še prekopirali na za to določeno spletno stran, predstavitev pa eno uro pred seminarjem na [http://bio.ijs.si/~zajec/poslji/ strežnik].<br /> * Vsi seminarji so v elektronski obliki dostopni [http://bio.ijs.si/~zajec/poslji/bioseminar/ tukaj].<br /> * Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo XY minut. Recenzenti morajo biti na predstavitvi prisotni.<br /> * Predstavitvi sledi razprava. Recenzenta morate predlagati vsaj eno izboljšavo predstavljenega projekta.<br /> <br /> ==&lt;font color=green&gt;Imena datotek&lt;/font&gt;==<br /> Prosim vas, da vse datoteke poimenujete po naslednjem modelu:<br /> * 20_nano_Priimek.doc za seminar, npr. 20_nano_Craik_Venter.doc<br /> * 20_nano_Priimek.ppt za prezentacijo, npr. 20_nano_Craik_Venter.ppt<br /> <br /> ==Urejanje spletnih strani na wikiju==<br /> Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''<br /> <br /> ==Citiranje virov==<br /> Citiranje je možno po več shemah, važno je, da se držite ene same. V seminarskih nalogah in diplomskih nalogah FKKT uprabljajte shemo citiranja, ki je pobarvana &lt;font color=green&gt;zeleno&lt;/font&gt;.<br /> Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.<br /> Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati.&lt;br&gt;<br /> '''Citiranje knjig:'''&lt;br&gt;<br /> Priimek, I. ''Naslov''. Kraj: Založba, letnica.&lt;br&gt;<br /> Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN.&lt;br&gt;<br /> <br /> Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005.&lt;br&gt;<br /> Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4.&lt;br&gt;<br /> Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.). <br /> <br /> '''Citiranje člankov:'''&lt;br&gt;<br /> Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani.&lt;br&gt;<br /> &lt;font color=green&gt;Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., Smith, H. O. in Venter, J. C.<br /> Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, 317, str. 632-638.&lt;/font&gt;<br /> <br /> Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole:&lt;br&gt;<br /> Priimek, I. (letnica, številka). Naslov. Naslov revije, strani.&lt;br&gt;<br /> Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.<br /> <br /> Revija Science uporablja skrajšani zapis:&lt;br&gt;<br /> C. Lartigue ''et al''. Science 317, 632 (2007)&lt;br&gt;<br /> <br /> V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne). Navesti morate tudi vse avtorje dela, razen v primeru, ko jih je 10 ali več. Takrat navedite le prvih devet, za ostale pa uporabite okrajšavo in sod. (in sodelavci). Pred zadnjim avtorjem naj bo vedno besedica &quot;in&quot;. <br /> <br /> <br /> '''Citiranje spletnih virov:'''&lt;br&gt;<br /> Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov&lt;br&gt;<br /> strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life&lt;br&gt;<br /> Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.</div>

Paulahorvat https://wiki.fkkt.uni-lj.si/index.php?title=BNT-seminar&diff=17928 2021-03-11T22:53:36Z

<p>Paulahorvat: </p> <hr /> <div>= Bionanotehnologija 2021- seminar =<br /> doc. dr. Gregor Gunčar, K2.022<br /> <br /> == Seznam seminarjev ==<br /> <br /> {| {{table}}<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''Vpisna številka'''<br /> 30170005 &lt;br /&gt;<br /> 30019058 &lt;br /&gt;<br /> 30200303 &lt;br /&gt;<br /> 30019363 &lt;br /&gt;<br /> 30019057 &lt;br /&gt;<br /> 30170131 &lt;br /&gt;<br /> 30170078 &lt;br /&gt;<br /> 30019040 &lt;br /&gt;<br /> 30170177 &lt;br /&gt;<br /> 30200324 &lt;br /&gt;<br /> 30019063 &lt;br /&gt;<br /> 30170103 &lt;br /&gt;<br /> 30170002 &lt;br /&gt;<br /> 30200319 &lt;br /&gt;<br /> 30200309 &lt;br /&gt;<br /> 30200320 &lt;br /&gt;<br /> 30019056 &lt;br /&gt;<br /> 30200311 &lt;br /&gt;<br /> 30200306 &lt;br /&gt;<br /> 30170243 &lt;br /&gt;<br /> 30019051 &lt;br /&gt;<br /> 30170141 &lt;br /&gt;<br /> 30170061 &lt;br /&gt;<br /> 30019035 &lt;br /&gt;<br /> 30200316 &lt;br /&gt;<br /> 30170222 &lt;br /&gt;<br /> 30200317 &lt;br /&gt;<br /> 30170193 &lt;br /&gt;<br /> 30200315 &lt;br /&gt;<br /> 30200307 &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''datum predstavitve'''<br /> 16.3.2021 &lt;br /&gt;<br /> 16.3.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 30.3.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 20.4.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 23.3.2021 &lt;br /&gt;<br /> 20.4.2020 &lt;br /&gt;<br /> 6.4.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 4.5.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> 30.3.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 11.5.2021 &lt;br /&gt;<br /> 13.4.2021 &lt;br /&gt;<br /> 18.5.2021 &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''recenzent1'''<br /> Anamarija Agnič &lt;br /&gt;<br /> Aljaž Bratina &lt;br /&gt;<br /> Urban Hribar &lt;br /&gt;<br /> Sabina Sladič Oblak &lt;br /&gt;<br /> Barbara Slapnik &lt;br /&gt;<br /> Tina Kolenc Milavec &lt;br /&gt;<br /> Klementina Polanec &lt;br /&gt;<br /> Ajda Godec &lt;br /&gt;<br /> Martin Špendl &lt;br /&gt;<br /> Tjaša Mlakar &lt;br /&gt;<br /> Sara Laznik &lt;br /&gt;<br /> Martina Lokar &lt;br /&gt;<br /> Doroteja Armič &lt;br /&gt;<br /> Martin Špendl &lt;br /&gt;<br /> Saša Slabe &lt;br /&gt;<br /> Mateja Žvipelj &lt;br /&gt;<br /> Špela Supej &lt;br /&gt;<br /> Urška Fajdiga &lt;br /&gt;<br /> Ernestina Lavrih &lt;br /&gt;<br /> Nika Mikulič Vernik &lt;br /&gt;<br /> Liza Ulčakar &lt;br /&gt;<br /> Urša Štrancar &lt;br /&gt;<br /> Tadej Medved &lt;br /&gt;<br /> Urška Zagorc &lt;br /&gt;<br /> Nina Lukančič &lt;br /&gt;<br /> Urška Pečarič Strnad &lt;br /&gt;<br /> Anže Karlek &lt;br /&gt;<br /> Luka Gnidovec &lt;br /&gt;<br /> Urša Lovše &lt;br /&gt;<br /> Almina Tahirović &lt;br /&gt;<br /> | align=&quot;center&quot; style=&quot;background:#f0f0f0;&quot;|'''recenzent2'''<br /> Urška Pečarič Strnad &lt;br /&gt;<br /> Urška Zagorc &lt;br /&gt;<br /> Liza Ulčakar &lt;br /&gt;<br /> Urša Štrancar &lt;br /&gt;<br /> Sara Laznik &lt;br /&gt;<br /> Luka Gnidovec &lt;br /&gt;<br /> Irma Zeljković &lt;br /&gt;<br /> Eva Keber &lt;br /&gt;<br /> Jernej Imperl &lt;br /&gt;<br /> Barbara Slapnik &lt;br /&gt;<br /> Urša Lovše &lt;br /&gt;<br /> Mateja Žvipelj &lt;br /&gt;<br /> Neža Pavko &lt;br /&gt;<br /> Almina Tahirović &lt;br /&gt;<br /> Nika Mikulič Vernik &lt;br /&gt;<br /> Urška Fajdiga &lt;br /&gt;<br /> Saša Slabe &lt;br /&gt;<br /> Klementina Polanec &lt;br /&gt;<br /> Tjaša Mlakar &lt;br /&gt;<br /> Ajda Godec &lt;br /&gt;<br /> Jernej Imperl &lt;br /&gt;<br /> Špela Supej &lt;br /&gt;<br /> Aljaž Bratina &lt;br /&gt;<br /> Nina Lukančič &lt;br /&gt;<br /> Matija Ruparčič &lt;br /&gt;<br /> Anže Karlek &lt;br /&gt;<br /> Sabina S. Oblak &lt;br /&gt;<br /> Urban Hribar &lt;br /&gt;<br /> Mirsad Mešić &lt;br /&gt;<br /> Tina Kolenc Milavec &lt;br /&gt;<br /> <br /> ==Naloga==<br /> Pripravite projektno nalogo iz področja Bionanotehnologije. Najpomembnejša je originalna ideja za nek izvedljiv projekt, ki pa mora biti takšen, da pritegne investitorje. Ker je pomembno tudi kako boste to naredili, morate predstaviti tudi metodo in ne samo ideje. Natančno morate vedeti, kako boste projekt izvedli.<br /> <br /> Predlagana struktura teksta:<br /> * Uvod<br /> * Predstavitev problema, znanstvena izhodišča, cilji<br /> * Izvedba projekta, metodologija, tehnike, materiali, vprašanja, hipoteze<br /> * Literatura<br /> <br /> Za pripravo seminarja velja naslednje:&lt;br&gt;<br /> * Elektronska verzija seminarja: avtor, naslov projekta, razširjeni povzetek projekta- 350-400 besed (brez literature) in grafični povzetek (čez približno pol strani). Vse naj bo na maksimalno dveh straneh, a ne sme vsebovati manj kot 350 besed (sem se ne šteje literatura). <br /> * Elektronsko verzijo seminarja oddajte en dan pred predstavitvijo, kasneje pa boste vsebino še prekopirali na za to določeno spletno stran, predstavitev pa eno uro pred seminarjem na [http://bio.ijs.si/~zajec/poslji/ strežnik].<br /> * Vsi seminarji so v elektronski obliki dostopni [http://bio.ijs.si/~zajec/poslji/bioseminar/ tukaj].<br /> * Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo XY minut. Recenzenti morajo biti na predstavitvi prisotni.<br /> * Predstavitvi sledi razprava. Recenzenta morate predlagati vsaj eno izboljšavo predstavljenega projekta.<br /> <br /> ==&lt;font color=green&gt;Imena datotek&lt;/font&gt;==<br /> Prosim vas, da vse datoteke poimenujete po naslednjem modelu:<br /> * 20_nano_Priimek.doc za seminar, npr. 20_nano_Craik_Venter.doc<br /> * 20_nano_Priimek.ppt za prezentacijo, npr. 20_nano_Craik_Venter.ppt<br /> <br /> ==Urejanje spletnih strani na wikiju==<br /> Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''<br /> <br /> ==Citiranje virov==<br /> Citiranje je možno po več shemah, važno je, da se držite ene same. V seminarskih nalogah in diplomskih nalogah FKKT uprabljajte shemo citiranja, ki je pobarvana &lt;font color=green&gt;zeleno&lt;/font&gt;.<br /> Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.<br /> Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati.&lt;br&gt;<br /> '''Citiranje knjig:'''&lt;br&gt;<br /> Priimek, I. ''Naslov''. Kraj: Založba, letnica.&lt;br&gt;<br /> Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN.&lt;br&gt;<br /> <br /> Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005.&lt;br&gt;<br /> Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4.&lt;br&gt;<br /> Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.). <br /> <br /> '''Citiranje člankov:'''&lt;br&gt;<br /> Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani.&lt;br&gt;<br /> &lt;font color=green&gt;Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., Smith, H. O. in Venter, J. C.<br /> Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, 317, str. 632-638.&lt;/font&gt;<br /> <br /> Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole:&lt;br&gt;<br /> Priimek, I. (letnica, številka). Naslov. Naslov revije, strani.&lt;br&gt;<br /> Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.<br /> <br /> Revija Science uporablja skrajšani zapis:&lt;br&gt;<br /> C. Lartigue ''et al''. Science 317, 632 (2007)&lt;br&gt;<br /> <br /> V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne). Navesti morate tudi vse avtorje dela, razen v primeru, ko jih je 10 ali več. Takrat navedite le prvih devet, za ostale pa uporabite okrajšavo in sod. (in sodelavci). Pred zadnjim avtorjem naj bo vedno besedica &quot;in&quot;. <br /> <br /> <br /> '''Citiranje spletnih virov:'''&lt;br&gt;<br /> Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov&lt;br&gt;<br /> strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life&lt;br&gt;<br /> Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.</div>

Paulahorvat