Alveolar echinococcosis (AE), a severe and life-threatening disease is caused by the small fox tapeworm Echinococcus multilocularis. Currently, the options of chemotherapeutic treatment are very limited and are based on benzimidazole compounds, which act merely parasitostatic in vivo and often display strong side effects. Therefore, new therapeutic drugs and targets are urgently needed. In the present work the role of two evolutionarily conserved signalling pathways in E. multilocularis, namely the insulin signalling cascade and Abl kinases, has been studied in regard to host-parasite interaction and the possible use in anti-AE chemotherapy. / Die alveoläre Echinokokkose ist eine ernste und lebensgefährliche Erkrankung, die durch den kleinen Fuchsbandwurm ausgelo ̈st wird. Die gegenwärtigen chemotherapeutischen Behandlungsmöglichkeiten beschränken sich auf die Behandlung mit Benzimidazolen, die in vivo nur parasitostatische Wirkung besitzen und häufig sehr starke Nebenwirkungen aufweisen. Aus diesem Grund besteht ein dringendes Bedürfnis nach neuen Medikamenten und Angriffszielen für diese. In der vorliegenden Arbeit wurde die Rolle zweier evolutionsgeschichtlich konservierter Signalsysteme, der Insulin Signalweg und die Abl Kinasen in E. multilocularis in Hinblick auf die Wirt-Parasiten Interaktion und dem mo ̈glichen Nutzen in der AE Chemotherapie untersucht.

Insulin ist ein essentielles Hormon im menschlichen Körper, welches für die Senkung der Blutglukosekonzentration, die Bildung von Energiespeichern und das Zellwachstum verantwortlich ist. Eine mit der Fehlregulation der Insulinproduktion einhergehenden Krankheit ist der Diabetes mellitus. Für diese Arbeit spielt der Typ 2 dieser Erkrankung eine wichtige Rolle. Es entwickelt sich bei Patienten mit diesem Typ des Diabetes mellitus langsam eine Insulinresistenz, die zunächst durch eine kompensatorische Überproduktion von Insulin charakterisiert ist. Dieser Zustand der Hyperinsulinämie kann Jahre bis Jahrzehnte andauern, ehe es zu einem Versagen der ß-Zellen des Pankreas und somit zu einer Hypoinsulinämie kommt. In dieser Arbeit war es Ziel herauszufinden, ob diese lange Zeit herrschende Hyperinsulinämie einen Einfluss auf die menschliche DNA hat. Die Genotoxizität von hohen Insulinkonzentrationen wurde in Hep-G2 Zellen, HT29 Zellen, sowie primären humanen peripheren Lymphozyten mithilfe des Comet Assays und des Mikrokerntests nachgewiesen. Oxidativer Stress bzw. dessen Reduzierung durch Antioxidantien und Inhibitoren wurde in HT29 Zellen mithilfe der DHE-Färbung detektiert. Diese Arbeit belegt dass sich Insulin schädigend auf das menschliche Genom in vitro auswirken kann. Eine besondere Relevanz haben die durchgeführten Experimente mit primären menschlichen Lymphozyten. Denn bei ihnen handelt es sich um Zellen, die im Gegensatz zu der auch genutzten humanen Leberkarzinomzelllinie Hep-G2 und der humanen Kolonkarzinomzelllinie HT29 nicht transformiert sind. Eine weitere wesentliche Erkenntnis dieser Arbeit ist, dass schon pathophysiologisch vorliegende Insulinkonzentrationen in der Lage sind Genomschädigungen in vitro zu induzieren. HT29 Zellen zeigten bei Kurzzeitbehandlung mit nur 1nM Insulin eine signifikante Erhöhung der DNA-Schädigung. Bei Langzeitexposition von 6 Tagen konnten schon 0,5nM signifikante DNA-Schäden hervorrufen. Diese durch Insulin hervorgerufenen Schäden könnten, falls sie so auch in vivo entstehen, bei Versagen von Reparaturmechanismen zur Entstehung von Mutationen und sich daraus entwickelnden Karzinomen beitragen. Aus diesem Grund war ein weiteres Ziel dieser Arbeit herauszufinden, ob bestimmte Antioxidantien oder Inhibitoren in der Lage sind die Insulin-induzierten Genomschädigungen zu verringern. Hierfür wurde Tempol, Apocynin, Plumbagin, VAS2870, Rotenone, PPP, HNMPA-(AM)3 und Wortmannin genutzt. Tatsächlich sind diese Substanzen in der Lage die durch Insulin hervorgerufene Schädigung zu reduzieren. Die positiven Ergebnisse dieser Arbeit könnten einen ersten Hinweis auf eine mögliche pharmakologische Intervention bei Hyperinsulinämie mit dem Ziel der Senkung des erhöhten Krebsrisikos geben. Eine wichtige Erkenntnis aus den Ergebnissen meiner Arbeit ist, dass die Reduzierung des oxidativen Stresses eine Reduzierung der Genomschädigung bewirkt. Die genutzten Substanzen Apocynin, Tempol, VAS2870 und Rotenone bewirkten in HT29 Zellen eine signifikante Reduzierung des durch Insulin ausgelösten oxidativen Stresses. Um aber genauere Aussagen über Möglichkeiten der Therapie bei Hyperinsulinämie zu treffen, sollten Folgestudien auch in vivo folgen, welche die in dieser Arbeit beschriebenen Effekte bestätigen. / Insulin is an essential hormone in the human body, which is responsible for the reduction of blood glucose concentration, the formation of energy holding compounds, and cell growth. A disease associated with the dysregulation of insulin production is diabetes mellitus. For this work, the type 2 of the disease plays an important role. Patients with this type of diabetes mellitus gradually develop an insulin resistance, which is initially characterized by a compensatory overproduction of insulin. This state of hyperinsulinemia may last years or even decades, before it comes to a break-down of the beta cells of the pancreas, and hence to a hypoinsulinemia. The target of this study was to evaluate, whether these long term lasting hyperinsulinemia has an impact on human DNA. The genotoxicity of high insulin concentrations was detected in HepG2 cells, HT29 cells as well as primary human peripheral lymphocytes, using the comet assay and the micronucleus test. Oxidative stress and it´s reduction by antioxidants and inhibitors was detected in HT29 cells using the DHE staining. This work shows that insulin may damage the human genome in vitro. A special relevance show experiments carried out with primary human lymphocytes. Because these are not transformed like the human liver carcinoma cell line HepG2, and the human colon carcinoma cell line HT29, also studied. Another significant finding of this study is, that even pathophysiologically present insulin concentrations are capable to damage the genome in vitro. HT29 cells showed a significant increase in DNA damage when short-term treated with only 1nM insulin. After long-term exposure of 6 days already 0.5nM were capable to significantly damage DNA. This induced insulin damage could, if also occurring in vivo, contribute to the development of mutations and carcinomas, if mechanisms of repairs are failing. A further aim of this study was therefore to check whether certain antioxidants or inhibitors would be capable of reducing insulin-induced genome damages. For this Tempol, Apocynin, Plumbagin, VAS2870, Rotenone, PPP, HNMPA-(AM)3 and Wortmannin were used. In fact, these substances are capable to reduce the damage caused by insulin. The positive results of this work could be a first indication of a possible pharmacological intervention against hyperinsulinemia with the aim of lowering stringent cancer risk. An important conclusion of the results of my work is that reduction of oxidative stress results in a reduction of genome damage. The substances used, Apocynin, Tempol, VAS2870 and Rotenone, caused a significant reduction of insulin-induced oxidative stress, in HT29 cells. In order to make more detailed statements about possibilities of hyperinsulinemia therapy, follow-up studies in vivo are required which would back-up the findings of this study.

Prior to the commencement of this study in 1990, a number of reports had appeared in the literature describing the importance of insulin action during lactation in mammals (see Chapter 1). These studies investigated the changes in circulating insulin and glucagon concentrations during lactation, the relative numbers of insulin receptors in insulin-sensitive tissues, and glucose utilisation by these tissues. However, at that time, no information was available on the structure of the mammary insulin receptor. The rationale for undertaking this study was to characterise the structure of the rat mammary insulin receptor as a means of furthering our understanding of the role insulin plays during lactation.An initial requirement of this study was the development of a method for the convenient and inexpensive preparation of A14-tyrosyl[125I]iodoinsulin. A14-tyrosyl[125I]iodoinsulin displays binding characteristics which are virtually indistinguishable from the native hormone, which is a necessary requirement for tracers which are to be used in binding studies. In Chapter 2, I describe a method for the purification of A14-tyrosyl[125I]iodoinsulin from a mixture of iodinated insulin molecules which are produced following oxidation by chloramine-T in the presence of Na125iodine. In this method I employed disposable cartridges packed with a C18 support matrix to which the iodinated insulin molecules are readily adsorbed when in an aqueous solution.A 14-tyrosyl[125I]iodoinsulin absorbed most strongly to the C18 matrix and unwanted products were removed through a sequence of washes prior to the elution of the A14-tyrosyl[125I]iodoinsulin derivative using a buffer containing 50% (v/v) acetonitrile. This prodct was unambiguously shown to be A14-tyrosyl[125I]iodoinsulin by N-terminal amino acid sequencing. The quality of this radiolabel compared favourably with commercially ++ / available A14-tyrosyl[125I]iodoinsulin preparations both in terms of specific activity and stability upon storage at -20C. Furthermore, a modified method based on this protocol has been used in our and other laboratories for the isolation of other iodinated peptides with highly satisfactory results.I have established that the size of the a-subunit of the rat mammary insulin receptor is significantly diminished compared with the liver insulin receptor (125 kDa versus 130 kDa). This difference in size was present throughout all stages of lactation and was not due to proteolysis of a larger form. Furthermore, I demonstrated that both the mammary and liver insulin receptor a-subunits migrated equally on PAGE following treatment with neuraminidase, indicating that the apparent size difference may be accounted for by a variation in the extent of receptor sialation. Treatment of the mammary insulin receptor a-subunit with glycopeptidase F demonstrated that the size of the aglycoreceptor (100 kDa) was similar to that described for insulin receptors from other insulin-sensitive tissues.I characterised the distribution of mRNA encoding the two, naturally-occurring insulin receptor isoforms in mammary tissue throughout all stages of pregnancy and lactation. These insulin receptor isoforms differ due to the absence (IR-A) or presence (IR-B) of a 12 amino acid peptide, encoded by exon 11 of the insulin receptor gene, and located near the C-terminus of the insulin receptor a-subunit. Mammary tissue predominantly expressed IR-A mRNA in contrast to liver tissue, which almost exclusively expressed IRB mRNA. Furthermore, the ratio of IR-A to IR-B mRNA in mammary tissue changed significantly during the first week post-partum whilst the distribution of IR-A and IR-B mRNA in the liver remained constant throughout pregnancy and lactation. This difference in insulin receptor isoform ++ / expression between mammary and liver tissue also contributed to the estimated size difference between the insulin receptor a-subunits from these two tissues. In addition, I characterised the expression of IR-A and IR-B mRNA in several different tissues obtained from rats on day 14 of gestation through to 7 days post partum. I established that the splicing mechanism is functional at least as early as day 14 of gestation, suggesting a possible role for the preferential expression of a particular insulin receptor isoform during organogenesis. I observed that IR-A mRNA was the predominant isoform in all foetal tissue studied, and the proportion of this isoform declined as the animal matured. These changes were significant in cardiac muscle, kidney and most dramatic in the liver where the expression of IR-A mRNA changed from 53% in the 21 day old foetus (the day before parturition) to 13% in the 1 day old neonate. These results suggest that the splicing mechanism which generates the receptor isoforms is subject to acute hormonal and/or metabolic control.The current literature suggests that the carbohydrate moieties of the insulin receptor affects its affinity for insulin. Furthermore, the IR-A and IR-B isoforms have been shown to display a 2-fold difference in their insulin binding affinity when expressed in heterologous cell lines such at CHO cells or Rat-1 fibroblasts. Since both glycosylational and isoform distribution differences were evident between mammary and liver tissues, the insulin binding affinities of these receptors were compared. Estimates of the binding affinity parameters were performed at both 4 C and 37 C. At both temperatures the equilibrium binding constants for mammary and liver tissues were not significantly different suggesting that structural variations of the mammary insulin receptor had no effect on the insulin binding affinity under the ++ / conditions described in this study. Comparison of the 4 C and 37 C binding data showed that the mammary insulin receptor exhibited complex, temperature-dependent binding characteristics, similar to those previously described for the liver insulin receptor, and entirely consistent with the presence of a temperature-dependent regulatory protein that affects insulin binding.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 118-125).

Residual feed intake (RFI) is one method to identify feed efficient animals; however, this method is costly and time consuming therefore, identifying an indirect measure of RFI is important. Evaluating the glucoregulatory mechanisms in cattle selected for divergent RFI may provide insight into metabolic processes involved in feed efficiency. This study evaluated the effect of a glucose (GLUC) challenge on efficient (LRFI) and inefficient (HRFI) tropically adapted bulls and heifers. Insulin (INS) secretion was determined by radioimmunoassay (RIA) and GLUC was determined by colorimetry. Insulinogenic index (IIND) was calculated as the ratio of INS to GLUC (I/G). Bonsmara heifers were evaluated in two experiments. Similar results were observed in both experiments. RFI affected (P < 0.05) INS response; with LRFI heifers having a greater INS response than HRFI heifers. Similarly, RFI affected (P < 0.05) IIND; with LRFI heifers having a greater IIND than HRFI heifers. In Santa Gertrudis bulls, RFI did not affect (P > 0.05) GLUC conc. or Ins. response; however, numerically HRFI bulls had a greater INS response than LRFI bulls. RFI affected (P < 0.05) IIND; with LRFI bulls having a lower IIND than HRFI bulls. In Brahman heifers (Exp 1), RFI did not affect (P > 0.05) GLUC concentration or INS. response; however, numerically HRFI heifers had a greater INS response than LRFI heifers. RFI affected (P < 0.05) IIND; with LRFI heifers having a lower IIND than HRFI heifers. In Brahman bulls (Exp 2), RFI affected (P > 0.05) INS response; with HRFI bulls having a greater INS response than LRFI bulls. RFI affected (P < 0.05) IIND; with LRFI bulls having a lower IIND than HRFI bulls. Bonsmara cattle evaluated for RFI had a response to an influx of exongenous glucose that was opposite to that observed in the Brahman and Santa Gertrudis cattle evaluated for RFI. Insulinogenic index was significantly different between RFI groups in each experiment. The lower amount of INS required for clearance of the GLUC from the circulation of the Brahman and Santa Gertrudis cattle fits with our hypothesis that more efficient cattle would require less INS than the less efficient cattle. Further research and studies need to establish glucoregulatory differences between breeds and sexes of cattle evaluated for RFI.

Syfte: Att sammanställa vilken forskning det finns kring relationen mellan fysiskt aktivitet och insulindoser samt blodsockervärden hos personer med diabetes typ 1. Metod: Litteraturstudie. En artikelsökning gjordes i databaserna PubMed och CINAHL. Sökningen resulterade i 17 studier. Efter granskning och analys av studierna synliggjordes två kategorier som redovisas i resultatet. Resultat: Flera studier kom fram till att fysisk aktivitet sänker blodsockervärdena hos personer med diabetes typ 1. En studie belyste att fysisk aktivitet måste pågå regelbundet för att resultat skall ses på lång sikt. Några studier belyste att medelintensiv träning sänker blodsockervärdena mer än högintensiv träning. Fem studier fann ett samband mellan fysisk aktivitet och behov av mindre insulindoser. Några studier upptäckte inte något samband mellan fysisk aktivitet och sänkta blodsockervärden. Om fysisk aktivitet är kopplat till bättre eller sämre glykemisk kontroll är studierna oense om. Viktigt är att mängden insulin anpassas efter den fysiska aktiviteten. Slutsats: Ett flertal av studierna visade att fysisk aktivitet sänker blodsockervärdena i blodet. För att då uppnå glykemisk kontroll behöver insulindosen justeras. Det är nödvändigt att det ges rekommendationer gällande både fysisk aktivitet och insulinmängd baserat på individen och inte ett generellt antagande.

We study the conformational change of insulin fibril growth from three aspects: the impact of (i) incubation time; (ⅱ) nano-particles; (iii) and ion added. We used circular dichroism (CD) spectroscopy and fourier transform infrared spectroscopy (FT-IR) to obtain the structural transition of the insulin, and gain the morphology information of fibril by atomic force microscopy (AFM) and transmission electron microscopy (TEM). We show that the insulin transform from £\-helix to £]-sheet structure as increased incubated time. The addition of Au nanoparticles (NPs) caused the formation of coordination bond with insulin fiber and produced shorter and thicker insulin fibril . The Fe3O4 NPs, on the other hand, offered only van der Waals interaction toward insulin fibril. Hence they could be used to separate insulin fibril from solution. Finally, addition of salts can induce the conformation changes of insulin fibril ten times faster than that without salts. And the insulin fibril fragment was two or three times shorter than that produced without salts. At high salt concentration, insulin formed amorphous aggregates. This phenomenon was attribute to anions from salt: covering the surface charge of insulin fibril, they weaken the original electrostatic repulsion among insulin fibrils and result in their aggregation.

In this report, electrostatic force microscopy (EFM), zeta-potential analyzer, circular dichroism Spectrophotometer and Fourier transform infrared spectroscopy are used to study the electrostatic property of surface on insulin fibril. EFM provides a simultaneous probe of topography and electrostatic property with highly local resolution in nanometer-scale. To understand the correlation between charge distribution on fibril surface and structure transformation, we controlled the incubation time and salt concentration. The results show that the surface charge increases with incubation time, and with the salt concentration increased, the pitch is found to decrease, variation of charge distribution are increased. In addition, we also have evaluated influence of amino acid sequence on growth rate of fibril associated with bovine, porcine and human insulin by atomic force microscopy and circular dichroism Spectrophotometer. The experimental results show that the number of hydrophilic groups on the peptide sequence will affect the speed of fibril generated, and the location of hydrophilic groups on the peptide sequence will affect the stability of the fibril under the frozen environment.

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 67-84).