Magdeburg, Universiẗat, Diss., 2004. / Erscheinungsjahr an der Haupttitelstelle: 2003.

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Primary cilia are essential in brain development, as mediators of sonic hedgehog signaling. However, their role in mature neurons remains elusive. One means to elucidate their function may be to investigate the function of the somatostatin type 3 receptor (SstR3), which is concentrated on the primary cilia of neurons. The inhibitory and anticonvulsant properties of somatostatin suggest that ciliary SstR3 might protect neurons against excitotoxicity, as seen in epileptic seizures. C57BL/6 wild type (wt) and SstR3 knockout mice were administered vehicle or epileptogenic agents kainic acid (KA) or pentylenetetrazole. Seizure behaviors were rated on seizure severity scales. KA-induced seizure behaviors were more severe in SstR3 mutants than in wt. Correspondingly, the mutants showed greater reactive gliosis, as indicated by increased numbers of GFAP immunoreactive (GFAP(+)) astrocyte processes. In addition, seizure severity was associated with a greater percentage of neural stem cells having an ACIII(+) cilium. Following injections of pentylenetetrazole, SstR3 mutants reached maximum seizure levels faster than wt. These results support the hypothesis that ciliary SstR3 are neuroprotective in mature neurons, and may provide a new avenue for the treatment of seizures.

Somatostatin is a neuropeptide found throughout the brain. Several studies have established its anatomical distribution as being quite heterogenous with relatively high concentrations appearing in the limbic and striatal systems. Presently, very little is known about the functions of somatostatin systems in the brain and how they interact with other transmitter systems. The following report is a summary of experiments undertaken to assess the functional and chemical interactions of somatostatin with other neurotransmitter systems. Previous studies have established that the dopaminergic inputs to the basal ganglia are important for locomotor activity and reward. These systems have also been implicated in several mental and neural diseases such as schizophrenia, depression, and Parkinson’s disease. In the first experiment, interactions between dopamine and somatostatin systems were examined using paradigms involving behavioural responses to dopamine agonists. Depletion of somatostatin levels by the drug cysteamine was found to attenuate amphetamine- and apomorphine-mediated motor behaviours but not the reinforcing aspects of amphetamine. The second experiment attempted to further characterize the nature of the dopamine-somatostatin interaction by examining the effects of haloperidol, a dopamine antagonist, on central somatostatin levels. Short term treatment with haloperidol decreased striatal somatostatin levels. Long term treatment (8 months) with haloperidol failed to alter somatostatin levels in the caudate-putamen. Since somatostatin levels appear to be normal in Parkinsonian brains, the effects of MPTP poisoning in mice on central somatostatin levels was also studied to examine the accuracy of this animal model of Parkinson's disease and examine the effects of dopaminergic lesions on somatostatin levels. The results of this experiment indicate that MPTP causes a dose dependent increase in nigral somatostatin levels without altering striatal or cortical levels. These results are in partial disagreement with results obtained from both post-mortem Parkinsonian brains and primates given MPTP, thereby questioning the accuracy of this mouse model of Parkinson's disease. The final experiment examined the effects of the anticonvulsant-antidepressant carbamazepine on central somatostatin levels in the rat. Although the chemical mechanisms responsible for the therapeutic effects of carbamazepine are unknown, previous studies have suggested that its efficacy in the treatment of both manic-depression and epilepsy may be associated with the ability of this drug to reduce the abnormal somatostatin levels observed in these diseases. In this experiment, neither acute, chronic, nor withdrawal from chronic treatment with carbamazepine were found to alter the levels of somatostatin in rats. The lack of effects of carbamazepine on basal somatostatin levels may indicate somatostatin cells are susceptible to carbamazepine only under pathological situations. Together, these results are discussed in the context of recent observations of abnormal somatostatin levels in several diseases of the central nervous system and provide some insight into the interactions and functions of somatostatin systems in the normal and abnormal brain. / Medicine, Faculty of / Graduate

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu). / Liver is considered the main glucostatic organ in the mammals. However, under a variety of physiological and pathological conditions the kidney plays a significant role in controlling glucose homeostasis. Regulation of renal gluconeogenesis by peptide hormones has not been extensively studied. Somatostatin, the growth hormone release inhibiting factor, has been shown to stimulate renal gluconeogenesis. In this study the detail mechanism of somatostatinstimulated renal glucose production is investigated. Prior treatment of the animals with reserpine to deplete tissues catecholamine stores did not abolish the stimulatory effact of soma tost a tin indicating that catecholamine rel ease may not mediate the enhanced gluconeogenic activity. Somatostatin effect was blocked by the alpha1-antagonist, prazocin, but not by the alpha2 antagonist, yohimbine, suggesting that alpha1 adrenergic receptors may be involved in somatostatin action. somatostatin decreased glucagonstimulated cyclic AMP accumulation and caused small but significant increase in renal cyclic AMP levels. It is proposed that somatostatin may act as a partial agonist to stimulate cyclic AMP production in rat renal tissues. Somatostatin-stimulated renal glucose synthesis is calcium dependent, since in a calcium-free system, somatostatin had no effect. Furthermore, somatostatin increased 45cCa++_ influx into renal tissues. The key rate limiting gluconeogenie reactions is stimulated by somatostatin in the renal cells as demonstrated by the increase in incorporation of [14c] from [14c]-pyruvate and [14c]-bicarbonate into glucose. In addition, somatostatin infusion increased the activities of the key enzymes, phosphoenol pyruvate carboxykinase and pyruvate carboxylase with no effect on fructose 1,6-bisphosphatase nor glucose-6-phosphatase. Somatostatin is shown to bind to one class of recognition sites in the rat renal plasma membranes which showed high Na-K ATPase and adenylate cylase (positive marker enzymes) activites and low activities for succinic dehydrogenase, glucose-6-phosphatase and beta-glucuronidase (negative marker enzymes). The dissociation constant (Kd) for the binding was 0.91 ± 0.06 nM and the binding capacity (Bmax) was 37.59 ± 1.04 fmole/mg protein. Somatostatin and Tyr1-somatostatin displaced [125I]-Tyr1-somatostatin binding with inhibition constant (Kr) values of 31.5 and 100 pM, respectively. This indicates the presence of specific receptors for somatostatin on rat renal cells.

In der vorliegenden retrospektiven Analyse wurden die Daten von 66 Patienten, die an einem NET erkrankt und in der Universitätsmedizin Göttingen behandelt wurden, bezüglich der Ef-fekte der Therapie mit SAA statistisch aufgearbeitet, ausgewertet und die erhaltenen Daten anhand der aktuellen Literatur diskutiert. Ziel war es, die in vorherigen Studien beobachtete verlängerte Dauer der stable disease unter der Therapie mit SAA auch in unserem Patientenkollektiv nachzuweisen. Ein zusätzliches Au-genmerk lag auf weiteren Effekten der Therapie mit SAA: Verlauf der Tumormarker und der Symptomatik sowie Verlauf der Tumorgröße und der Metastasen, auch im Vergleich von SAA-Monotherapie vs. SAA plus Zusatztherapie. Dazu wurden die Daten der Patienten be-züglich oben genannter Aspekte ausgewertet und mit der Therapie in Beziehung gesetzt. Ne-ben anamnestischen Angaben und Laborwerten dienten hierfür auch Daten aus der Bildge-bung mittels CT, MRT und Ultraschall. Für die Aspekte der Symptomkontrolle, der Kontrolle der Tumormarker sowie des Tumorwachstums und der Entstehung von Metastasen konnte eine Überlegenheit des Zeitraums mit SAA gegenüber dem Zeitraum ohne SAA nachgewiesen werden. Der lange Beobachtungszeitraum und die dadurch nicht unerhebliche Anzahl von Patienten, die während der Analyse ausschieden, sowie die mit den Jahren veränderte und verbesserte Diagnostik (vor allem im Bereich der Tumormarker) und das Fehlen standardisier-ter Zeitintervalle bezüglich der Verlaufskontrolle sind jedoch als Einschränkung dieser Analy-se zu nennen. Im beobachteten Zeitraum vom 1. Januar 1975 bis 31. August 2011 ergab sich eine im Ver-gleich zu vorherigen Studien recht lange mittlere Beobachtungsdauer von 102 Monaten, in denen im Schnitt eine Monotherapie mit SAA über 28 Tage und eine Gesamtdauer der Thera-pie mit SAA über 47 Monate nachgewiesen werden konnte. Dieser von uns gezeigte und im Vergleich zu anderen, oben genannten Studien sehr lange Beobachtungszeitraum und das da-mit verbundene gute Langzeitüberleben lassen auf eine Proliferationshemmung und somit auf eine lange Dauer der stable disease schließen. Bezüglich der Symptomatik, die historisch ge-sehen die wichtigste Indikation zu einer solchen Therapie darstellt, konnte in unserem Kollek-tiv bei 17,8 % der Patienten (n = 8) eine Verbesserung unter SAA beobachtet werden. Die Werte der Tumormarker blieben unter einer Therapie mit SAA in 30,3 % der Fälle (n = 20) stabil. Die durchschnittliche Dauer der stable disease unter SAA betrug in unserer Analyse 21 Monate. Des Weiteren zeigt sich in unserem Kollektiv kein Unterschied in der Dauer der stab-le disease bezüglich des initialen Metastasierungsstadiums oder der Anwendung von zusätzli-chen Therapien. Im Größenverlauf des Primärtumors konnte bei 47 % der Patienten (n = 31) eine stable disease beobachtet werden. Wie schon in vorausgegangenen Studien beschrieben, bestätigen auch unsere Daten, dass zwischen der Dauer der stable disease und dem Verlauf der Tumormarker und der Symptomatik keine Korrelation besteht. Es zeigte sich eine Über-einstimmung von Wachstum des Primärtumors und Ausmaß der systemischen Ausbreitung, wobei Daten unserer Analyse zeigen, dass die Therapie mit SAA einen stärkeren Effekt auf den Verlauf des Primärtumors als auf den Verlauf bereits bestehender Metastasen hat. Anhand der in unserer Auswertung erzielten Ergebnisse kann gezeigt werden, dass die Be-obachtungen in unserem Patientenkollektiv mit denen der aktuellen Literatur vergleichbar sind. Festzuhalten bleibt somit, dass eine Therapie mit SAA den Krankheitsverlauf eines NET im Sinne einer stable disease um bis zu 21 Monate verzögern kann. Im besten Falle führt eine solche Therapie innerhalb dieses Zeitraums zusätzlich zu einer Besserung der klinischen Symp-tomatik. Eine Größenregredienz sowohl des Primärtumors als auch der Metastasen, wie sie auch nur vereinzelt in den früheren Studien beschrieben worden waren, konnte jedoch in unse-rem Patientenkollektiv nicht beobachtet werden. Dies unterstreicht die Aussage, dass es sich bei SAA um ein antiproliferatives, aber nicht zytoreduktives Therapiekonzept handelt. Studien, die neue Substanzen zur Behandlung von NETs zum Teil mit bereits großem Erfolg getestet haben, verdeutlichen die Suche nach einer immer effektiveren und individuelleren Therapie dieser Erkrankung. Langzeitergebnisse diesbezüglich bleiben jedoch abzuwarten.

Lai-Han Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 120-129). / Abstracts in English and Chinese. / Members of Thesis Advisory Committee --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / Abbreviations --- p.viii / Table of contents --- p.ix / Chapter Ch 1 --- General Introduction / Chapter 1.1 --- Growth hormone --- p.1 / Chapter 1.1.1 --- Physiology of GH --- p.1 / Chapter 1.1.2 --- Biological Functions of growth hormone --- p.2 / Chapter 1.1.3 --- Structure of growth hormone --- p.3 / Chapter 1.1.4 --- The physiological activities of growth hormone --- p.5 / Chapter 1.1.5 --- The importance of increasing grass carp production yield --- p.6 / Chapter 1.1.6 --- Application of recombinant growth hormone --- p.7 / Chapter 1.1.7 --- Application of recombinant grass carp GH --- p.8 / Chapter 1.2 --- Administration of GH to fish --- p.9 / Chapter 1.2.1 --- Methods in the administration of growth hormone to fish --- p.9 / Chapter 1.3 --- Different approaches in the expression of GH --- p.12 / Chapter 1.3.1 --- Bacterial expression system --- p.12 / Chapter 1.3.2 --- Baculovirus and yeast expression system --- p.14 / Chapter 1.4 --- Expression of grass carp growth hormone in yeast (Saccharomyces cerevisiae & Pichia Pastoris) --- p.14 / Chapter 1.4.1 --- Saccharomyces cerevisiae is widely used in the past --- p.14 / Chapter 1.4.2 --- Pichia pastoris is an alternative approach to overcome drawbacks of Saccharomyces cerevisiae --- p.16 / Chapter 1.5 --- Methods for increasing expression level of a cloned gene --- p.20 / Chapter 1.6 --- Purpose of present study --- p.22 / Chapter Ch 2 --- Materials and Method / Chapter 2.1 --- Bacterial strains --- p.23 / Chapter 2.2 --- Yeast strains --- p.23 / Chapter 2.3 --- Plasmids --- p.23 / Chapter 2.4 --- Bacterial culture Media and Solutions --- p.23 / Chapter 2.5 --- Antibiotic Solutions --- p.24 / Chapter 2.6 --- Restriction endonucleases and other enzymes --- p.24 / Chapter 2.7 --- Culture stock solutions --- p.24 / Chapter 2.8 --- SDS-PAGE and Western blot reagents --- p.28 / Chapter 2.9 --- DNA agarose gel --- p.30 / Chapter 2.10 --- General Techniques --- p.32 / Chapter 2.11 --- Construction of recombinant yeast strain --- p.40 / Chapter 2.12 --- Expression of the recombinant protein --- p.42 / Chapter 2.13 --- Purification of the protein --- p.44 / Chapter 2.14 --- Characterization of the protein --- p.46 / Chapter Ch3 --- Results / Chapter 3.1 --- Construction of Recombinant Plasmids --- p.50 / Chapter 3.2 --- Sequencing of the recombinants --- p.61 / Chapter 3.3 --- Linearization and electroporation of recombinant plasmids --- p.69 / Chapter 3.4 --- Optimization of electroporation condition of producing recombinant yeast cells --- p.70 / Chapter 3.5 --- Screening and selection of AOX characteristics (By MM plates) --- p.71 / Chapter 3.6 --- Screening and selection of AOX characteristics (By PCR) --- p.72 / Chapter 3.7 --- Selection of high expression clones --- p.77 / Chapter 3.8 --- Time course expression studies --- p.82 / Chapter 3.9 --- Batch fermentation --- p.89 / Chapter 3.10 --- Purification of r-gcGH --- p.92 / Chapter 3.11 --- Characterization of r-gcGH --- p.100 / Chapter 3.11.1 --- Immunological property of r-gcGH --- p.100 / Chapter 3.11.2 --- Biological activity of r-gcGH --- p.101 / Chapter Ch4 --- Discussion / Chapter 4.1 --- Evaluation of expression profile --- p.108 / Chapter 4.1.1 --- Expression yield --- p.108 / Chapter 4.1.2 --- Efficiency in the cleavage of signal peptide --- p.111 / Chapter 4.2 --- Evaluation of the biological activity of r-gcGH --- p.113 / Chapter 4.3 --- Evaluation of the post-translational modification of r-gcGH --- p.115 / Chapter 4.4 --- Further studies --- p.116 / Reference --- p.120

Universiẗat, Diss., 2005--Würzburg.

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