Background: Type 2 diabetes mellitus is a metabolic disorder characterized by the rise of fasting plasma glucose from its normal range (≥125mg/dl). It is marked by insufficient production of insulin from pancreatic β-cells as a result of failed compensation due to insulin resistance. Several treatments are available for the disorder, which mainly focus on improving the sensitivity of insulin in different body tissues. Recently, probiotics were suggested as candidate treatments for type 2 diabetes and for extending lifespan as well. This experiment aims to investigate such claims using Drosophila melanogaster as a disease model.   Results: Other than the observed low average weights in treated larva samples, probiotics did not show any other significant results in affecting the length, glucose, glycogen, and trehalose levels (One-Way ANOVA and Kruskal-Wallis, p>0.05). Real-time PCR was only carried out once. Thus, no statistical tests were reliable enough to analyse the data obtained. The longevity study, on the other hand, did show significance (Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test, p<0.0001), as the probiotic Bifidobacterium lactis extended the lifespan of adult flies feeding on a high sugar diet significantly when compared to the control ones feeding on only high sugar diet without probiotics.   Conclusion: Except for weight measurements, none of the other results was reliable enough to make a concrete conclusion on whether the treatments indeed worked in reversing type 2 diabetes symptoms or not. Real-time PCR results did show some effects of some of the treatments at different developmental stages. However, unless Real-time PCR is repeated at least once using the same protocol, no deduction can be made. Additionally, the data obtained hint that the dosage used (0.025 g) was too high for larvae and adult flies and might have caused malnutrition by blocking their midgut and decreasing food absorption. Hence, false significant or non-significant results were acquired instead.   Further studies are required using a much lower probiotic dosage if Drosophila is used as a disease model. Although, other models such as mice or rats are recommended in this case, in order to reach a solid conclusion about the effectiveness of probiotics in treating type 2 diabetes mellitus. Baring these thoughts in mind and based on the results of this experiment, the null hypothesis indicating that there is no significant relationship between the use of probiotics and reversing type 2 diabetes mellitus symptoms is therefore accepted.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20523号 / 生博第365号 / 新制||生||48(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 松本 智裕, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle.

Prostate cancer is the most commonly diagnosed non-skin cancer in men and the second leading cause of cancer death in the United States. Prostate cancer, like many cancers, is a disease that generally requires a long period of time to develop and grow before it becomes detectable. This long period of latency makes prostate cancer a candidate for dietary chemoprevention. Soy and selenium (Se), are associated with a decreased risk of prostate cancer. We previously showed that high dietary intake of selenium (Se) and soy isoflavones decreased the expression of the androgen receptor (AR) and AR-regulated genes in the prostates of healthy rats. In this study we hypothesized that the downregulation of AR and AR-regulated genes would inhibit tumorigenesis in the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse. Mice were fed one of two stock diets with or without a supplement of Se in a 2 X 2 factorial design. The stock diets provided high or low dietary isoflavones. Mice were exposed to the diets from conception and sacrificed at 18 or 24 weeks of age. Prostate histopathology, urogenital tract (UGT) weight, serum IGF-1 levels, and the expression of AR and AR-regulated genes in the dorsolateral prostate was examined using quantitative PCR and Western blotting. Urogenital tract (UGT) weight was reduced compared to control in all dietary groups containing high Se, isoflavones, or both at 24 weeks (p<0.005). Dietary isoflavones delayed tumor progression and downregulated protein levels of AR, AR-regulated genes, and upregulated the protective FOXO1 and FOXO3a transcription factors. High dietary isoflavones also decreased the phosphorylation of the IGF-1R. The only main effect of Se was the upregulation of AKR1C14 the enzyme that deactivates 5&aplha;-DHT.This study identifies a previously unknown effect of isoflavones in the upregulation of FOXO expression and confirms previous studies of isoflavones' anticancer effects. Further research is needed to find a protective dose or form of Se and to elucidate the mechanism of isoflavones.

<p>Upon activation mast cells release numerous proinflammatory mediators. With this feature, mast cells play an important role in host defense against pathogens, and are involved in tissue remodeling and wound healing. However, in cases of excessive inflammation the effects of mast cells are detrimental. This is observed in allergy, asthma, rheumatoid arthritis, atherosclerosis, certain types of heart failure, and in several other chronic destructive inflammations. Mast cell numbers are typically increased at inflammatory sites. There they act both directly, as effector cells, and in a regulatory manner, secreting agents that recruit and activate other immune cells.</p><p>The studies presented here investigated mechanisms regulating mast cell numbers at sites of inflammation, focusing on cell migration and regulation of survival/apoptosis. We report that SCF-induced mast cell migration requires p38 MAP kinase activity. Moreover, we found that SCF-mediated mast cell survival is regulated through downregulation of the proapoptotic Bcl-2 family member Bim, as well as through phoshorylation of Bim. SCF seems to control Bim protein levels via FOXO transcription factors, and to induce phosphorylation of Bim via the Mek/Erk and the PI3-kinase/Akt signaling pathways. Furthermore, mast cell death triggered by deprivation of SCF and/or IL-3 involves the Bim protein, as demonstrated using <i>bim</i>-/- mast cells. Additional studies revealed that IgE-receptor activation, which occurs in allergy, promotes both prosurvival and proapoptotic signaling events. This includes upregulation of Bim and the prosurvival Bcl-X_L and A1, as well as phosphorylation of Akt, FOXO factors, GSK-3β, IκB-α, Bad, and Bim. The simultaneous stimulation of prosurvival and proapoptotic signaling events could be a way to fine-tune the fate of mast cells after IgE-receptor activation and degranulation.</p><p>The new insights about mechanisms involved in mast cell migration and regulation of survival/apoptosis might prove useful for future efforts to design new drugs to be used for mast cell-associated diseases.</p>

Upon activation mast cells release numerous proinflammatory mediators. With this feature, mast cells play an important role in host defense against pathogens, and are involved in tissue remodeling and wound healing. However, in cases of excessive inflammation the effects of mast cells are detrimental. This is observed in allergy, asthma, rheumatoid arthritis, atherosclerosis, certain types of heart failure, and in several other chronic destructive inflammations. Mast cell numbers are typically increased at inflammatory sites. There they act both directly, as effector cells, and in a regulatory manner, secreting agents that recruit and activate other immune cells. The studies presented here investigated mechanisms regulating mast cell numbers at sites of inflammation, focusing on cell migration and regulation of survival/apoptosis. We report that SCF-induced mast cell migration requires p38 MAP kinase activity. Moreover, we found that SCF-mediated mast cell survival is regulated through downregulation of the proapoptotic Bcl-2 family member Bim, as well as through phoshorylation of Bim. SCF seems to control Bim protein levels via FOXO transcription factors, and to induce phosphorylation of Bim via the Mek/Erk and the PI3-kinase/Akt signaling pathways. Furthermore, mast cell death triggered by deprivation of SCF and/or IL-3 involves the Bim protein, as demonstrated using bim-/- mast cells. Additional studies revealed that IgE-receptor activation, which occurs in allergy, promotes both prosurvival and proapoptotic signaling events. This includes upregulation of Bim and the prosurvival Bcl-XL and A1, as well as phosphorylation of Akt, FOXO factors, GSK-3β, IκB-α, Bad, and Bim. The simultaneous stimulation of prosurvival and proapoptotic signaling events could be a way to fine-tune the fate of mast cells after IgE-receptor activation and degranulation. The new insights about mechanisms involved in mast cell migration and regulation of survival/apoptosis might prove useful for future efforts to design new drugs to be used for mast cell-associated diseases.

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Die Herzinsuffizienz ist aufgrund ihrer weltweit ansteigenden Prävalenz und den damit einhergehenden beträchtlichen Kosten im stationären sowie ambulanten Sektor von großer Relevanz für die globalen Gesundheitssysteme. Trotz multipler Möglichkeiten der pharmakologischen und chirurgischen Therapie ist die Mortalität weiterhin hoch. Die Etablierung neuer medikamentöser Behandlungsansätze ist daher von enormer Bedeutung und bedarf der Erweiterung des Verständnisses der pathophysiologischen Grundlagen der Erkrankung. Kardiomyopathien zählen zu den häufigsten Ursachen der Herzinsuffizienz. Dysfunktionen des Ubiquitin-Proteasom-Systems (UPS) wurden bereits in der Progression der Herzinsuffizienz bei Kardiomyopathien diskutiert und setzten einen potentiellen Fokus auf die E3-Ligasen. Diese Hypothese aufgreifend hatte die vorliegende Arbeit das Ziel, erstmalig den kompletten Myostatin/AKT/forkhead box protein (FOXO)-Signalweg und dessen Auswirkungen auf die E3-Ligasen muscle atrophy F-box gene (MAFbx) und muscle ring-finger protein-1 (MuRF1) im fortgeschrittenen humanen Kardiomyopathie-Stadium zu charakterisieren und mögliche neue Ansatzpunkte für die pharmakologische Therapie verschiedener Formen der Kardiomyopathie zu ermitteln. Zu diesem Zweck wurden Myokardproben von 26 Patienten mit ischämischer Kardiomyopathie (ICM), 23 Patienten mit dilatativer Kardiomyopathie (DCM) und 17 Kontrollpatienten molekularbiologisch, proteinbiochemisch und immunhistochemisch analysiert, um Veränderungen der Komponenten der Myostatin/AKT/FOXO-Signalkaskade und der E3-Ligasen MAFbx und MuRF1 zu identifizieren. Aus diesen Untersuchungen resultierten umfangreiche Alterationen des Signalwegs vor allem in DCM-Patienten. In dieser Gruppe war die Gen- und Proteinexpression der E3-Ligase MAFbx und des Transkriptionsfaktors FOXO1 gegenüber der Kontrollgruppe verringert. Weiterhin zeigte sich eine Reduktion von AKT auf der Genexpressionsebene und von Myostatin auf der Proteinebene in der DCM-Gruppe. Die ICM-Patienten wiesen mit Reduktionen der Genexpression von AKT und MAFbx sowie einem verringerten Anteil MuRF1-positiver Zellen in den immunhistochemischen Analysen nur geringe Unterschiede gegenüber der Kontrollgruppe auf. Die limitierten Veränderungen des Myostatin/AKT/FOXO-Signalwegs und der E3-Ligasen MAFbx und MuRF1 in ICM-Patienten lassen auf eine eher begrenzte Relevanz der Signalkaskade in der Pathophysiologie dieser Erkrankung schließen. Im Kontrast dazu stehen die detektierten Alterationen der analysierten Zielmoleküle in der DCM-Gruppe. Diese Divergenzen zeigen Unterschiede in der Pathogenese von ICM und DCM auf der Ebene des UPS. Daher unterstützt diese Arbeit die Hypothese, dass eine Therapieoptimierung dieser beiden Kardiomyopathien durch pharmakologische Adressierung verschiedener molekularer Ansatzpunkte erreicht werden könnte. In dieser Arbeit konnte die simultane Reduktion von MAFbx und FOXO1 in DCM-Patienten gezeigt werden. Durch die Validierung dieser synergistischen Regulation könnte FOXO1 einen potentiellen Angriffspunkt neuer medikamentöser Therapien der DCM darstellen. Unklar bleibt, ob die beobachtete Reduktion von MAFbx Ausdruck eines kardialen Schutzmechanismus oder der Progression des pathologischen Remodelings ist. Daher müssen weiterführende Studien klären, ob die E3-Ligase MAFbx über die therapeutische Beeinflussung des Transkriptionsfaktors FOXO1 herauf- oder herabreguliert werden sollte. Die Ergebnisse der vorliegenden Arbeit geben zudem einen Hinweis darauf, dass die Regulation von FOXO1 und MAFbx in DCM-Patienten nicht allein von Myostatin und AKT abhängig ist. Die Betrachtung weiterer Einflussfaktoren von MAFbx und FOXO1 in humanem Herzmuskelgewebe könnte somit ebenfalls Gegenstand zukünftiger Forschungsvorhaben sein. / Disturbances in the ubiquitin proteasome system, and especially changes of the E3 ligases, are subjects of interest when searching for causes and therapies for cardiomyopathies. The aim of this study was to clarify whether the myostatin/AKT/forkhead box O (FOXO) pathway, which regulates the expression of the E3 ligases muscle atrophy F-box gene (MAFbx) and muscle ring-finger protein-1 (MuRF1), is changed in dilated cardiomyopathy of ischemic origin (IDCM) and dilated cardiomyopathy of non-ischemic origin (NIDCM). The mRNA and protein expression of myostatin, AKT, FOXO1, FOXO3, MAFbx and MuRF1 were quantified by real-time polymerase chain reaction and ELISA, respectively, in myocardial tissue from 26 IDCM and 23 NIDCM patients. Septal tissue from 17 patients undergoing Morrow resection served as a control. MAFbx and FOXO1 mRNA and protein expression (all p < 0.05), AKT mRNA (p < 0.01) and myostatin protein expression (p = 0.02) were decreased in NIDCM patients compared to the control group. Apart from decreases of AKT and MAFbx mRNA expression (both p < 0.01), no significant differences were detected in IDCM patients compared to the control group. Our results demonstrate that the myostatin/AKT/FOXO pathway is altered in NIDCM but not in IDCM patients. FOXO1 seems to be an important drug target for regulating the expression of MAFbx in NIDCM patients.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20779号 / 生博第385号 / 新制||生||51(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 米原 伸, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM