Dissertations / Theses on the topic 'INS-1 cells'

The pathophysiology of cytokines released by infiltrating white blood cells upon pancreatic beta cells is not fully understood. Early growth response gene-1 (Egr-1) expression is specifically and transiently up regulated in pancreatic beta cells in response to glucose. We hypothesized that interleukin-1 beta (IL-1▀) induction of nitric oxide alters glucose induced Egr-1 transcription levels. Egr-1 levels were assessed via western blot, nitric oxide was measured with a Griess Reagent kit and insulin levels via ELISA. Glucose induced both insulin and Egr-1 production in INS-1 cells. IL-1▀ dose dependently increased nitric oxide production over time and significantly attenuated glucose induced Egr-1 expression. Sodium nitroprusside dose dependently reduced glucose induced Egr-1 production. The data suggest a strong relationship between IL-1▀ induced nitric oxide production and the reduction of glucose stimulated Egr-1 production. The pathways altered by this cytokine could provide a better understanding of the pathophysiology leading to pancreatic beta cell death.

L’Ins(1,4,5)P3 joue un rôle évident dans la signalisation cellulaire :il permet la libération du Ca 2+ des stocks intracellulaires par son action au niveau de récepteurs spécifiques. Pour mettre fin à son action, l’Ins(1,4,5)P3 peut être dégradé par une Ins(1,4,5)P3 5-phosphatase en Ins(1,4)P2, un métabolite inactif. L’Ins(1,4,5)P3 peut aussi être transformé en Ins(1,3,4,5)P4 par une Ins(1,4,5)P3 3-kinase. L’Ins(1,3,4,5)P4 semble posséder des capacités de signalisation propres ou au contraire liées à celles de l’Ins(1,4,5)P3.

L’Ins(1,3,4,5)P4 est aussi le point de départ de toute une série d’inositol hautement phosphorylés, dont les rôles ne sont pas clairs. Trois isoformes de l’Ins(1,4,5)P3 3-kinase existent (A, B et C). Ces isoformes possèdent un domaine catalytique carboxy-terminal bien conservé. Par contre, les domaines amino-terminaux sont spécifiques et leur permettraient d’établir des interactions ou de subir des régulations propres. Pour tenter d’élucider le rôle fonctionnel de l’Ins(1,3,4,5)P4, nous avons généré et analysé des souris déficientes pour les isoformes B et C de cette enzyme.

Les souris déficientes pour l’Ins(1,4,5)P3 3-kinase C ne présentent pas de phénotype évident, ce qui suggère que son rôle n’est pas crucial ou que son absence peut être compensée par une autre enzyme.

Les souris déficientes pour l’Ins(1,4,5)P3 3-kinase B, par contre, présentent une immunodéficience caractérisée par une absence spécifique des lymphocytes T αβ périphériques. Cette absence fait suite à un blocage dans la différenciation du précurseur du lymphocyte, le thymocyte. Les caractéristiques de la signalisation induite par le récepteur de surface (TCR) permettent la sélection des thymocytes, de manière à constituer un pool de lymphocytes T restreints pour le MHC et tolérants pour le soi. Nous avons montré que ces phénomènes de sélection étaient défectueux dans les thymocytes mutants, du fait de leur hyporéactivité à la stimulation par le TCR. Le mécanisme responsable de cette hyporéactivité n’est pas encore élucidé. A première vue, la mobilisation de Ca 2+ ne semble pas altérée dans ces thymocytes mutants en réponse à des stimulations classiques. Cependant, d’autres types de stimulation, se rapprochant plus de celles réellement rencontrées par le thymocyte in vivo, doivent encore être investigués. L’intégrité d’autres voies de signalisation cruciales du lymphocyte T doit aussi être vérifiée.

En conclusion, l’isoforme B de l’Ins(1,4,5)P3 3-kinase et l’Ins(1,3,4,5)P4 qu’il produit jouent un rôle crucial dans la différenciation du thymocyte, par un mécanisme qui reste encore à déterminer.
Doctorat en sciences biomédicales
info:eu-repo/semantics/nonPublished

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75.
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.

ABSTRACT Prostasin is expressed in normal prostate epithelial cells but down-regulated in prostate cancers, while prostasin re-expression in invasive prostate cancer cells reduced invasion. We examined prostasin expression and function in benign prostatic hyperplasia (BPH). We evaluated prostasin expression in 12 BPH specimens by immunohistochemistry, and evaluated the impact of prostasin silencing by siRNA on the expression of the inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), and cyclin D1, as well as on cell proliferation and invasion, using the BPH-1 human prostate epithelial cell line model. Prostasin expression was localized in the glands of BPH tissues by immunohistochemistry, in either the tall columnar-shaped or the flattened epithelial cells. We silenced prostasin expression by >50% at both the mRNA and protein levels using siRNA in the BPH-1 human prostate epithelial cell line, and this silencing of prostasin expression was associated with an induction of iNOS and ICAM-1 expression and a down-regulation of cyclin D1 expression. The protein expression of EGFR, a putative prostasin substrate, was not affected by prostasin silencing in this cell line. The prostasin-silenced cells displayed a reduced cell proliferation rate and reduced invasiveness, cell behaviors regulated by cyclin D1, iNOS, and ICAM-1 in the BPH-1 cells. We believe that this down-regulation of cyclin D1 is due to prostasin's augmentative effect on iNOS. We also believe that the decrease in cell motility is due to an increase in iNOS and ICAM-1 as well as a decrease in cyclin D1, since all of these molecules can play a role in cell motility. In conclusion, Prostasin is somehow involved in the regulation of inflammatory gene expression (iNOS and ICAM-1) in prostate epithelial cells, as well as cyclin D1 expression, cell proliferation and invasion, involving molecular mechanisms different than those in the prostate cancer cells. These studies suggest that prostasin is a player in the glandular components of benign prostatic hyperplasia.
M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS

The production of Nitric Oxide (NO) under physiological conditions has beneficial roles in acting as a key signaling component of many biological processes as well as having an anti-microbial effect. However its effects following excess production by the inducible NO pathway is potentially detrimental in the pathogenesis of chronic inflammation including sepsis and several other inflammatory diseases. Understanding the mechanisms that regulate the expression of the inducible nitric oxide synthase (iNOS) responsible for producing the excessive amounts of NO in disease states is therefore critical. In this regards, experiments were carried out to identify the signaling pathways that may mediate this process, focusing specifically on the JAK/STAT cascade. The reason for selecting the latter is because our research group, amongst others, has carried out extensive work investigating other signaling pathways, including the mitogen activated kinases (MAPK). Moreover, studies have also been carried out in an attempt to identify the critical role of JAK/STAT signaling for iNOS induction. These studies however failed to conclusively demonstrate whether, as with the MAPKs, the JAK/STATs may also play an essential role. Furthermore there is indeed controversy in the literature with researchers unable to agree whether expression of iNOS does require JAK/STAT activation. Thus, the aim of the project described in this thesis was to establish unequivocally whether activation of the JAK/STATs preceeds induction of iNOS. The studies were extended to L-arginine transport as well because the latter is widely reported to be induced in parallel with iNOS and substrate supply to iNOS may be critical for sustained NO production. Changes in transporter activity as well as their expression profiles were assessed. All experiments were carried out in either rat aortic smooth muscle cells (RASMCs) or in the J774 macrophage cell line. These cell types were selected because RASMCs are one of the prime targets for induced NO production in vascular inflammation and the macrophages are involved in host defence, acting in part through NO production. To establish the role of JAK/STATs, pharmacological and molecular approaches were used. Pharmacologically, two inhibitors were used and these were AG490 and JAK inhibitor I. The former is reported to be a selective JAK2 inhibitor and the other blocks all known JAK proteins. The potential of the GTPases to regulate the induction of iNOS was also examined using selective inhibitor known to regulate these proteins. In addition to these drugs, siRNA targeting JAK2 was also exploited and western blotting was extensively used to detect expression of various proteins including iNOS, native and phosphorylated JAK2 and TYK2. Changes in iNOS activity was monitored by determining nitrite production using the Griess assay and L-arginine transport was monitored using tritiated arginine (L-[3H]arginine). RASMCs were treated with a combination of LPS (100 µg/ml) and IFN- (100 U/ml) and the macrophages with LPS (1 µg/ml) to induce iNOS and transporter activity. Consistent with previous reports, the above treatment of both cell types resulted in the expression of iNOS, production of NO and enhanced transport of L-arginine. These effects were not affected by AG490 but blocked by JAK inhibitor I. Furthermore, although both cell types expressed the key JAKs (JAK2 and TYK2), neither of these proteins were phosphorylated under conditions of induced NO production. Moreover, siRNA experiments showed that JAK2 expression could be abolished without any significant change in NO production, confirming that at least JAK2 may not be required for this process. Whether TYK2 is involved still remains to be resolved as the phosphor-protein could not be detected. However the conclusive siRNA knockdown studies could not be carried out due to time and cost constraints. Apart from iNOS and NO production, changes in induced L-arginine transport were also not significantly affected under the experimental conditions described above suggesting that like with iNOS, induction of L-arginine transport is independent of at least JAK2. Interestingly however, STAT-1 was phosphorylated and this was blocked by JAK inhibitor I but not AG490. Thus, STAT-1 activation may be essential but its activation may be independent of the JAKs. One possible alternate upstream activator of STAT-1 may be the GTPases. Indeed these proteins have been indicated to phosphorylate STAT-1 independent of the JAKs. However, in this project, inhibition of the GTPase pathway enhanced NO production and L-arginine transport suggesting that the GTPases downregulate these processes. In conclusion, the studies carried out in this thesis have shown that induction of iNOS, NO production and L-arginine transport in both RASMCs and J774 macrophages are independent of JAK2 but require STAT-1 activation which may be phosphorylated independently of the JAKs. The role of other JAKs such as TYK2 although unlikely, will need to be resolved using a more specific approach such as siRNA.

It is becoming increasingly more common to use culture expanded human mesenchymal stem cells (hMSCs) in regenerative medicine due to their low incidence in vivo. However, their successful application is hampered by a lack of selective markers to positively identify the expanded multipotent cells. This study aimed to characterise STRO-1 antigen as a potential biomarker of multipotency on cultured bone marrow derived hMSCs. In an attempt to identify the nature of this antigen, two techniques were implemented: peptide phage display technology and a microarray based approach. Changes in the expression of STRO-1 were investigated during culture expansion of hMSCs. STRO-1 expression positively correlated with cellular morphology and multilineage potential, whereby senescent cells down-regulated STRO-1 antigen and exhibited decreased adipogenic and osteogenic potential. Furthermore, STRO-1 was found to be heterogeneously expressed on hMSC populations and enrichment followed by lineage specific induction of the STRO-1BRIGHT fraction resulted in enhanced adipogenic and osteogenic differentiation potential. The expression of STRO-1 antigen was further characterised as a marker of differentiation, whereby differentiating cells were found to down-regulate STRO-1. A cellular hierarchy in hMSC population was therefore proposed based on STRO-1 status, with the highest STRO-1 expressive cells representing the multipotent subset. In an attempt to identify the epitope that STRO-1 IgM antibody recognised, peptide phage display technology was used as solid and liquid phase panning systems but the approach yielded no promising peptide candidate. Subsequently, comparative gene expression microarray analysis of osteosarcoma cell lines (143B, CAL72, G-292, HOS, MG-63, Saos-2 and U-2-OS) was implemented and a list of eight potential candidate genes encoding STRO-1 antigen was selected. This work ultimately led to the identification of putative cancer stem cells (CSCs) in seven osteosarcoma cell lines initially based on STRO-1 expression. With MG-63 strongly expressing STRO-1, the maintenance of MSC-like properties by STRO-1 expressing cell lines was investigated. A heterogeneous pattern of osteogenic differentiation was observed between and within the cell lines. Closer inspection revealed a cellular hierarchy comprising of holoclones and paraclones, with the holoclones representing the putative CSCs in osteosarcoma. Overall, this thesis addressed the fields of regenerative medicine as well as oncology by proposing STRO-1 antigen as a marker of multipotency on hMSCs and osteosarcoma holoclonal cells as the putative cancer stem cell targets for anti-cancer drug development.

Die Infektion mit dem Erreger der Malaria tropica, Plasmodium falciparum, verläuft individuell unterschiedlich. Während manche der Infizierten rasch an einer komplizierten Malaria versterben, zeigen andere keinerlei Symptomatik, obwohl jahrelang eine Parasitämie besteht. Was diese Individuen voneinanderen unterscheidet, ist weitgehend unbekannt. Morbidität und Mortalität der Erkrankung sind von der Auseinandersetzung zwischen Wirt und Parasit abhängig, die von exogenen und endogenen Faktoren beeinflußt wird. Unter den endogenen Faktoren spielen die genetischen Determinanten, die sowohl an angeborenen als auch an erworbenen Resistenz- und Immunmechanismen beteiligt sind, eine besondere Rolle. In den hier zusammengefaßten Arbeiten wurden als Determinanten der angeborenen Resistenz gegenüber Malaria die Sichelzellanämie, Alpha-Thalassämie, G6PD-Mangel und der HLA-Klasse-II-Polymorphismus und als genetische Einflußfaktoren von erworbenen Immunmechanismen Varianten des TNF-Promotors, von ICAM-1 und iNOS untersucht. Die Arbeiten unterstützen die Hypothese, daß die Interaktion von Mensch und Plasmodien zu einer ständigen gegenseitigen Beeinflussung und Anpassung geführt hat. Die koevolutonäre Veränderung der Genome der beiden Organismen ist wahrscheinlich mitverantwortlich für die unterschiedliche geographische Verteilung von Genvarianten sowohl des Menschen als auch der Plasmodien und scheint auch heute noch Teil einer komplexen und dynamischen Anpassung von Wirt und Parasit zu sein.
The manifestation of an infection with Plasmodium falciparum, the pathogen of malaria, is individually different. Some indiviuals have a high risk of developing severe malaria, whereas others remain asymptomatic despite a long-lasting parasitemia. The basis of these differences is unknown. Morbidity and mortality of malaria are dependent on the interaction between the host and the parasite which is influenced by exogenic and endogenic factors. The latter are determined by genetic elements involved in innate and acquired mechanisms of resistance and immunity. The studies summerized here address genetic determinants of innate resistance against malaria (sickle cell trait, alpha-thalassemia, G6PD deficiency, blood groups and HLA class II alleles) and those of acquired immunity (variants of the TNF promoter, ICAM-1, and iNOS). The results support the view that the interaction between humans and plasmodia has led to continuous mutual influences and adaptations. Probably, the co-evolution of the genomes of both organisms is jointly responsible for the different geographical distribution of parasitic and human gene variants. This process seems to be part of an ongoing complex and dynamic adaptation of the host and the parasite.

IL-1 is a pro-inflammatory cytokine which play an important role in the activation and regulation of host defence and immune responses to inflammation or injury. IL-1 is able to bind and activate IL1-RI and IL1-RII, which are found on many cells types. The role of the IL-1 signalling in the deployment of Th cell subsets, especially Th17 cells is well known. However, the specific cells which are responsible for the expression of IL-1 signalling in the immune defense and in the development of the Th cell lineage in response to infection, is still largely unclear. Therefore in this thesis, IL1-RI conditional knockout mice specifically in hematopoietic cells (IL1-RI vaviCre+) were generated. Using IL1-RI vaviCre+ mice in comparison with IL1-RI global knockout mice (IL1-RI-/-) would determine whether the expression IL-1 signalling from hematopoietic cells is responsible for the immune defense and in the development of the Th1, Th2 and Th17 cells against gastrointestinal helminth Trichuris muris (T.muris) infections. The generation of IL1-RI vaviCre+ mice have been investigated at the genomic and proteomic level in order to confirm that the Il1-rI gene is inactivated in hematopoietic cells. The characterisation of IL1-RI vaviCre + mice at the genomic level confirmed that the Il1-rI gene was obliterated successfully. At protein level the characterisation of IL1- RI vaviCre + mice confirmed that IL1-RI was dysfunctional in hematopoietic cells. Additionally, the development of the immune cells was investigated in IL1-RI vaviCre + and IL1-RI-/- mice. Our findings demonstrated that the lymphocyte development was not affected by the deletion of the IL1- RI gene. This data indicated that IL1- RI vaviCre + and IL1-RI-/- mice are vital in vivo models. In high dose infection, both IL1-RI vaviCre + and IL1-RI -/- mice were able to clear the infections due to their ability to generate a Th2 response. Both IL1-RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris were susceptible to infections and showed high levels of Th1 cytokines. Thus, we hypothesised that IL1-RI signalling in hematopoietic cells was not required for worm expulsion and the generation of Th2 and Th1 response. Interestingly, low dose T.muris infection showed a clear reduction in the Th17 cytokines IL22 and IL17 in both IL1-RI vaviCre + and IL1-RI -/- mice, suggesting that IL-1 signalling expressed from hematopoietic cells is responsible for the development of Th17 cells and secretion of IL17 and IL22. IL1- RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris also showed an increase in inflammation in the colon and decreased of goblet cell hyperplasia. It is well known that IL22 plays an important role in preventing tissue damage and repair. Thus, in this study IL22 global knockout mice (IL22 -/-) were used to determine if the change in crypt lengths and goblet cell hyperplasia in IL1-RI vaviCre + and IL1-RI -/- was due to an absence of IL22. Our finding showed that IL22 -/- mice infected with low dose of T.muris had increased crypt length and a reduction in goblet cells. The similar phenotype in crypt length and goblet cell hyperplasia between IL22 -/-, IL1-RI vaviCre + and IL1-RI -/- mice suggested that a lack of IL22 in IL1-RI vaviCre + and IL1-RI -/- mice is responsible for the change in mice phenotype. It also provides more evidence for the role of IL-1 signaling in hematopoietic cells in the generation of Th17 cells and in the production of its cytokine IL22.IL1-RII is an inhibitor of IL1-RI, thus, in this study IL1-RII global knockout mice (IL1-RII -/-) mice was used in comparison with IL1-RI -/- mice to verify the role of IL-1 signaling in the development of Th17 cells. Our finding showed an overexpression of IL17 and IL22 in IL1-RII -/- compared with IL1-RI -/- mice and a higher level of IL17 in IL1-RII -/- mice compared with IL1-RII flox/flox mice. This data confirmed that IL-1 signaling is important for the development of Th17 cells and the production of its cytokine IL17 and IL22.

Type 1 diabetes (T1D) is an autoimmune disease, resulting from the specific destruction of the insulin‐producing beta‐cells in the islets of the pancreas. Islet‐specific autoreactive T cells are instrumental in this process and although these cells are present in individuals with and without T1D, their exhibition of a memory phenotype in diabetic individuals indicates they may have been previously activated in these patients. This suggests a breakdown in peripheral tolerance, implying regulatory T cells (Tregs) may be involved. Indeed, CD4+ CD25hi FOXP3+ Tregs do not differ in frequency in T1D, but their function is impeded. This defect is present in both recentonset type‐1 diabetics (ROT1D) and long‐standing type 1 diabetics (LST1D) suggesting it is a stable phenotype, possibly under genetic control. Also, it is known that the production and signalling of interleukin‐2 (IL‐2); a cytokine essential for the maintenance of Tregs, is defective in T1D. The aim of this thesis was to ascertain whether defective Treg function in T1D is genetically determined or a consequence of the disease. The first section of this thesis examined the effect of a T1D‐associated IL2RA single nucleotide polymorphism (SNP) on Treg function, by means of a genotype‐immunophenotype study. Non‐diabetic donors homozygous for the susceptible allele at this SNP exhibited diminished Treg fitness and suppressive action, suggesting that defective Treg function is a contributing factor in T1D. The latter section utilised T1D‐discordant monozygotic twins and healthy controls to examine Treg function and the IL‐2‐dependent generation of regulatory type 1 (Tr1) cells. Due to the low number of twins obtained nothing conclusive can be drawn from the study on Treg function. However, the results from the Tr1 generation assay suggest the possible existence of an IL‐2 signalling defect in non‐diabetic twins.

Angiogenesis is the process of new blood vessel sprouting from pre-existing vessels and is responsible for generating the majority of nascent vessels during development, tissue regeneration and disease. During angiogenesis, sprouting endothelial cells (ECs) are organised into leading 'tip' cells (TCs) and trailing 'stalk' cells (SCs). This hierarchal organisation of TCs and SCs is essential for the coordinated collective migration of ECs during sprouting. However, the precise mechanisms that define TC verses SC behaviour and identity remains uncertain. Transcriptomic analysis of sprouting vessels in zebrafish embryos led to the identification of a novel TC-associated gene, transmembrane 4 L six family member 1 (tm4sf1). We find that tm4sf1 expression is tightly spatiotemporally restricted to migrating TCs during intersegmental vessel (ISV) sprouting in zebrafish. Furthermore, TC tm4sf1 expression is controlled by the vascular endothelial growth factor receptor (Vegfr) - Notch signalling axis. Morpholino oligonucleotide (MO)-mediated knockdown of tm4sf1 reveals a subtle delay in ISV sprouting upon loss of tm4sf1 expression. Moreover, using multiplexed, real-time imaging approaches and in-depth analysis of TC and SC behaviours at single cell resolution, we reveal that the delay in ISV sprouting is specifically due to reduced TC motility. Furthermore, we find that tm4sf1 functions to induce TC motility in the leading daughter cell following TC mitosis, to rapidly re-establish post-mitotic TC behaviour. Generation of tm4sf1 mutant zebrafish lines using both transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) confirms that Tm4sf1 modulates TC behaviour. Additionally, mechanistic studies in human ECs reveal that tm4sf1 regulates VEGFR-mediated signalling upon VEGF-stimulation, which subsequently controls cell migration and expression of the TC determinants, DLL4 and VEGFR2. Hence, our results suggest that tm4sf1 is a novel modulator of the TC-SC hierarchy and collective EC migration during ISV sprouting. Overall, these findings have potential therapeutic implications since tm4sf1 may be a promising target for the manipulation of pathological angiogenesis in disease.

The goal of this research project is to understand how a high sugar diet may affect pancreatic beta cell function. High glucose concentrations lead to an increase in lipid droplets and TORC1 in beta cells, which promote high basal secretion of insulin (Erion K.A. et al., JBC, 2015). SREBP is a key regulator of cholesterol and lipid synthesis and depends on TORC1 activity. The active form of SREBP is located in the nucleus. Does glucose-induced lipid synthesis in beta cells increase via SREBP? To answer this question, we propose: 1) To test the effect of high glucose (11mM) on nuclear SREBP in INS-1 cells in comparison to physiological glucose (4mM). 2) To determine if nuclear SREBP is affected when PIP4Kgamma (a regulator of TORC1) is suppressed. SREBP translocation from the cytosol to the nucleus was measured by immunofluorescence. SREBP processing was measured by western blot. SREBP1 activation increased in response to prolonged exposure to excess glucose after at least 48hrs. Both translocation and processing increased in 11mM glucose compared to 4mM glucose. When PIP4Kgamma was suppressed in INS-1 cells, SREBP translocation was inhibited. Lipid droplet accumulation was measured by nile red staining and it was found that de novo lipid synthesis only contributes to a small fraction of total lipid droplets. In conclusion, SREBP is activated in beta cells when in excess glucose. This may allow for lipid accumulation and basal hypersecretion of insulin due to over nutrition.

OBJECTIVE: To investigate the effects of statin exposure on insulin secretion and metabolism in pancreatic beta cells in order to determine possible mechanisms that may contribute to the increased risk of developing Type 2 Diabetes that has been associated with taking statins. METHODS: Simvastatin and Pitavastatin were prepared to resemble the intracellular active form of the compound by converting the lactone, when present to a carboxyl group. These statins were then incubated at varying concentrations (0-200 nM) with clonal pancreatic β-cells (INS-1) for variable amounts of time (24-72 hours). Glucose-stimulated insulin secretion was measured from INS-1 cells cultured at low (4 mM) and high (11 mM) glucose with and without statin incubation. Results were measured using a homogenous time-resolved fluorescence (HTRF) insulin assay kit (Cisbio). The effect of statins on INS-1 cellular metabolism was determined by measuring oxygen consumption rate using a Seahorse XFe96 analyzer (Agilent Technologies). Statin effects on intracellular Ca2+ ([Ca2+]i) was examined in INS-1 cells cultured on glass bottom dishes loaded with the calcium indicator Fura-2 AM (Invitrogen) and mounted on an Olympus fluorescence microscope. RESULTS: Simvastatin significantly inhibited GSIS and depleted insulin content in a dose-dependent manner (25-200 nM) after 72-hour exposure. However, when normalized for insulin content, inhibition was not observed. In contrast, pitavastatin did not affect GSIS, but did decrease insulin content in a dose-dependent manner (25-200 nM). At both high and low glucose, simvastatin (200 nM) increased the frequency and amplitude of intracellular calcium ([Ca2+]) oscillations at 1, 3, and 12 mM glucose. Furthermore, simvastatin increased mitochondrial respiration at low glucose, in cells exposed to the highest dose of simvastatin (200 nM). CONCLUSION: Inhibition of GSIS by simvastatin and not pitavastatin, confirmed previous results from our lab that indicate that statins may have differential mechanistic effects on β-cells. Furthermore, intracellular calcium ([Ca2+])i imaging revealed that cells exposed to simvastatin (200 nM) had increased oscillation frequency and amplitude at basal (1, 3 mM) and high (12 mM) glucose conditions, indicating a potential difference in basal insulin secretion that may contribute to hyperinsulinemia. Lastly, mitochondrial respiration at low glucose increased in cells exposed to the highest dose of simvastatin (200 nM), suggesting that statins may play a role in decreasing energy efficiency.

OBJECTIVE: The aryl hydrocarbon receptor (AhR) translocates to the nucleus and binds to the aryl hydrocarbon receptor nuclear translocator (ARNT) to regulate biological responses upon ligand activation. The aim of this study was to measure the effects of activation or inhibition of AhR activity on basal and glucose-stimulated insulin secretion (GSIS) from clonal pancreatic β-cells (INS-1) cultured under normal and glucolipotoxic (GLT) conditions (high glucose and fatty acid). METHODS: Insulin content and secretion were measured utilizing homogenous time-resolved fluorescence (HTRF) insulin assay kit (cisbio). Cells cultured in RPMI media containing 5 mM and 11 mM glucose were pre-incubated with the receptor agonist FICZ or antagonist CH223191 for 96 hours. Insulin secretion over 2 hours was reported as ng/million cells. Intracellular lipid was measured by fluorescence after Nile red staining. RESULTS: Incubation of INS-1 cells with 11 mM glucose and fatty acid increased lipid droplets, basal insulin secretion and inhibited GSIS compared to cells cultured in 4 mM glucose, characteristic of GLT. Incubation of INS-1 cells with 11 mM glucose alone also exhibited GLT characteristics. INS-1 cells cultured at 11 mM glucose and treated with antagonist (1.25 - 10 μM) had decreased lipid content and improved insulin secretion compared to cells cultured in 11 mM glucose alone. INS-1 cells cultured in 5 mM glucose and treated with the AhR agonist (1.25 - 10 μM) exhibited increased intracellular lipid and impaired insulin secretion. CONCLUSION: The AhR may play a mediatory role in the development of GLT in pancreatic β-cells cultured in excess nutrients and β-cell specific activator or inhibitor ligands of this receptor could potentially be a targeted therapeutic treatment of diabetes.

OBJECTIVE: Cardiovascular disease (CVD) remains the leading cause of death globally. Reducing high blood cholesterol, which is a dominant risk factor for CVD events, is an essential goal of medical treatment. Statins are known as first‐choice agents. However, clinical trials report that some statins increased the risk for type 2 diabetes (T2D). Our objective was to investigate the effect of different statins on insulin secretion and content from pancreatic β-cells after chronic and acute exposure and determine the underlying mechanisms. METHODS: The effects of simvastatin, lovastatin and pitavastatin on GSIS and content were studied in clonal pancreatic β-cells (INS-1 832/13) cultured in high glucose (12 mM). Insulin content and secretion were measured after chronic and acute incubation of statins using homogenous time-resolved fluorescence (HTRF) insulin assay kit (Cisbio). Intracellular Ca2+ was measured using fura-2 AM (Invitrogen). RESULTS: Simvastatin (25-200 nM) and lovastatin (50-200 nM) significantly inhibited GSIS and depleted insulin content in a dose-dependent manner after 72-hour exposure. When the secretion level was normalized for content, the inhibitory effect was not observed. Simvastatin (200 nM) also increased the amplitude of intracellular Ca2+ oscillations at low glucose, but this was not reflected in the amplitude of oscillatory insulin release. In contrast, pitavastatin (25-200 nM) did not affect GSIS and only decreased insulin content at the highest dose tested. CONCLUSION: Inhibition of GSIS by simvastatin and lovastatin could be due to depletion of insulin content. Decreased Ca2+ sensitivity may also contribute to inhibition of GSIS by simvastatin. Pitavastatin had less inhibitory effect on GSIS and insulin content as compared to simvastatin and lovastatin indicating that not all lipophilic statins have a detrimental impact on GSIS. We suggest that statins may have differential mechanistic effects on β-cells some of which may contribute to the risk of T2D.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.

碩士
長庚大學
天然藥物研究所
93
Enterovirus 71 (EV71) is a widespread virus that causes severe and fatal diseases in patients, including circulation failure. The blood vessel will be damaged first , and produced necrosis and inflammation, this phenomenon is called vasculitis. Vasculitis is an inflammatory disorder mediated by vascular cell adhesion molecule-1 (VCAM-1) or nitric oxide. In previous study, the value of plasma levels of VCAM-1 and NO in the pathogenesis and prognosis was investigated in body with virus infections of varying severity. But the mechanisms underlying EV71-initiated intracellular signaling pathways to influence host cell functions remain unknown. In this study, we have identified EV71-indued PDGFR, Akt, ERK, p38, NF-B activation and iNOS, VCAM-1 expression in a time- and viral dose-dependent manner. Treatment of RV-SMCs with EV71 stimulated phosphorylation of PDGFR, Akt, ERK, and p38which was attenuated by AG1296, LY294002, Wortmannin, U0126, and SB202190, respectively. The PDGFR inhibitor (AG1296), PI3-K inhibitor (LY294002, Wortmannin), and ERK inhibitor (U0126) inhibited EV-71-induced iNOS expression. And the PDGFR inhibitor (AG1296), PI3-K inhibitor (LY294002, Wortmannin), p38 inhibitor (SB202190), and JNK inhibitor (SP600125) inhibited EV-71-induced VCAM-1 expression. Furthermore, iNOS and VCAM-1 expression induced by EV71 was significantly attenuated by a selective NF-B inhibitor (Helenalin). Consistently, EV71-stimulated translocation of NF-B into the nucleus and degradation of IB-α was blocked by Helenalin, AG1296, SB202190, SP600125, LY294002, and Wortmannin. These findings suggest that EV71 interacting through adhesion receptors initiates PDGFR-, PI3-K/Akt, p38-, and NF-B-dependent signaling pathways that mediate VCAM-1 expression in RV-SMCs, but not via p42/p44 MAPK. In addition, EV71 interacting through adhesion receptors initiates PDGFR-, PI3-K/Akt, p42/p44 MAPK-, and NF-κB-dependent signaling pathways that mediate iNOS expression in RV-SMCs.

Caenorhabditis elegans is a powerful tool to study cellular migration and morphogenesis during organ development. During pharynx development, the dorsal gland cell, g1p, is born in the anterior aspect of the pharyngeal primordium and undergoes a form of morphogenesis called retrograde extension. egl-15, the single Fibroblast Growth Factor Receptor (FGFR) in C. elegans and ina-1, one of two α-integrin receptors, are both required for the proper extension or migration of g1p cell. Mutations in either egl-15 or ina-1 show similar gland cell over-migration defects where the gland cell body migrates past the terminal bulb and is located in proximity of the intestine. The kinase domain of EGL-15 was found to be required for migration and transgenic rescue strategies were used to determine the tissue of EGL-15 function. RNA interference was used to determine if egl-15 and ina-1 are functioning in the same pathway to regulate gland cell migration.
February 2015

博士
國立交通大學
應用化學系碩博士班
100
Part 1: Deposition of InN Nanoparticles on TiO2 Films for Dye-Sensitized Solar Cell and Photoluminescene Studies The direct deposition of InN over TiO2 nanoparticle and nanotube (NP and NT) films was demonstrated by employing a plasma-enhanced chemical vapor deposition (PECVD) system with trimethyl indium (TMIn) and ammonia (NH3) as indium and nitrogen precursors, respectively. By varying the flow rate of TMIn at 358 K, enhancement in power conversion efficiencies by 4-23% and 1-11% under AM 1.5 illumination was observed for N3 dye sensitized solar cells using the InN deposited TiO2 NP and NT films, respectively. With a 9 μm thick TiO2 substrate, the most improvement of conversion efficiency increased from 6.57% to 8.20% by using 2 sccm of TMIn and 20 sccm of NH3 at 358 K for 10 min InN deposition on the TiO2 NP film. The enhancement by InN deposition can be explained by the improved absorption in the range of 400-500 nm and the increased loading of N3 dye molecules. This study describes the noticeable improvement of DSSC efficiencies by semiconductor nanoparticles deposited on TiO2 NP films. The enhancement of photoluminescence (PL) from anatase TiO2 thin films covered with InN nanoparticles by PECVD can also be observed. The strong PL band observed in the anatase TiO2 thin films with the coated InN may be attributed to the special interface of InN/O and/or more defects generated during the deposition. Part 2: Shock Tube Study on the Thermal Decomposition of Ethanol The thermal decomposition of C2H5OH highly diluted in Ar (1-100 ppm) has been studied by monitoring H atoms using the atomic resonance absorption spectrometry (ARAS) technique behind reflected shock waves over the temperature range 1308 - 1760 K at pressures: 1, 1.46 and 2 atm. Branching fractions for producing CH3+CH2OH (1a) and H2O+C2H4 (1b) have been examined by quantitative measurements of H atoms produced in the secondary decomposition of the product CH2OH; the pressure dependence of the branching fraction for channel (1a) is obtained by a linear least-squares analysis of the experimental data and can be expressed as f1a = (0.71±0.07) - (826±116)/T, (0.90±0.02) - (1079±34)/T, (1.02±0.10) - (1229±168)/T at P = 1, 1.46 and 2.0 atm, respectively, for T = 1450 - 1760 K. The rate constant obtained in this study is found to be consistent with previous theoretical and experimental results; however, the pressure dependence of the branching fraction obtained in this study is smaller than those of previous theoretical works. The experimental k1 and f1a can be summarized as follows: k1a/s-1 = F1a(6.07±0.18) x 10^10 exp[-(23850 ± 750)/T] k1b/s-1 = (1-F1a) (6.07±0.18) x 10^10 exp[-(23850 ± 750)/T] where, F1a is given by, F1a = (0.308P+0.420) – (399P+451)/T. The observed evolutions of [H], [CO] and [H2O] with time, together with the recent experimental and theoretical results on the kinetic parameters of the important related elementary reactions have been used to construct an extended reaction model for the pyrolysis of C2H5OH and compared with the previously proposed kinetic models.

碩士
國立中山大學
化學系研究所
106
In this research, we design new ratiometric fluorescent sensors based on semiconducting polymer nanoparticles (Pdots) due to the good photostability, large Stock shift and low bio-toxicity. Ratiometric measurement via FRET (Förster resonance energy transfer) mechanism have overcome some hindrance of nonratiometric methods such as the oscillation of concentration of probe and different excitation source. Herein, we demonstrated two specific-functional Pdots: one serves as near-infrared fluorescent probe for calcium ion detection and the other is a new kind of thermochromic anti-counterfeiting material. (1) Heptamethine Cyanine Dye Based Semiconducting Polymer Dots for Ratiometric Detection of Calcium Ions in Living Cells 　　Calcium ions are important in living systems and many biological processes can be monitored by approach fluctuations in calcium levels including helping blood clotting, cell membrane permeability control, anti-inflammatory action, maintenance of muscle motor contraction and detoxification. 　　Here we developed a Pdots-based probe conjugated with BAPTA coordination structure, extremely strong specificity for calcium ion, as calcium ion indicator. To enhance the signal-to-noise ratio, we doped heptamethine cyanine dye into polymer to achieve near-infrared fluorescence via FRET and integrated BAPTA chelator into backbone as NIR emission promoter via PET (Photoinduced electron transfer). Calcium ion could be effectively detected in vitro and in vivo by the fluorescence ratiometric measurement. Keywords : Semiconducting polymer dots、Near-infrared dyes、Calcium sensing、Photoinduced electron transfer、Fluorescence ratiometric analysis. (2) Fluorescent Inks Based on Thermo-responsive Semiconducting Pdots for Anti-counterfeiting Applications 　　Counterfeiting markets in currency, commodities, and medicines have led to serious economic losses and risks to the governments, while anti-counterfeiting technology could provide multiple layers of protection of these economic products. However, the rapid development of technology lead to the imitation of traditional anti-counterfeiting techniques (such as inks, watermarks, etc.). Therefore, a new type of anti-counterfeiting technology has become a necessary direction to combat a series of counterfeit products. 　　To surpass the afore-mentioned challenges, we combined Pdots with thermochromic modalities as a new type of dual colorimetric and fluorescent nanomaterial. Then, we tried to apply this thermochromic material on writing and printing for anti-counterfeiting intent. Keywords : Semiconducting polymer dots、Thermochromism、Microencapsulation、Dual Colorimetric、Anti-counterfeit technology.