Das primäre Offenwinkelglaukom (POWG) ist eine mit typischen Gesichtfeld- und Papillenschäden einhergehende Erkrankung des Auges, die in den westlichen Industrienationen zu den häufigsten Erblindungsursachen zählt. An Glaukom erkrankte Patienten weisen häufig erhöhte Augeninnendruckwerte und gesteigerte TGF-beta 2-Spiegel im Kammerwasser auf. Der Augeninnendruck wird im Wesentlichen durch den Abflusswiderstand des Trabekelmaschenwerks bestimmt. In der vorliegenden Arbeit wurde der Einfluss des Wachstumsfaktors TGF-beta 2 auf das Zytoskelett von menschlichen Trabekelmaschenwerkszellen (HTM) untersucht. Hierbei konnten die bereits bekannten TGF-beta-Effekte, nämlich verstärkte Stressfaserbildung und Zunahme der alpha-SMA- sowie Aktin-Expression bestätigt werden. Bisher unbekannt war die Zunahme der Expression von N-Cadherin und beta-Catenin unter TGF-beta, die Veränderungen der Zell-Zell-Adhäsionen nach sich zieht und damit auch Einfluss auf die biomechanischen Eigenschaften des Trabekelmaschenwerks haben könnte. beta-Catenin ist hierbei auch unter dem Einfluss von TGF-beta nur zu einem geringen Anteil im Zellkern lokalisiert, was mit einer vermehrten Lokalisation von beta-Catenin in Zell-Zell-Verbindungen vereinbar ist. Da vermutlich unter TGF-beta-Stimulation sogar eher weniger beta-Catenin als Mediator für den Wnt-Signalpfad zur Verfügung steht, könnte ein TGF-beta-vermittelter Wnt-Antagonismus eine Rolle in der Entstehung des POWG spielen. Es ist bekannt dass eine Hemmung des Wnt-Signalwegs den Augeninnendruck erhöht. Im Rahmen dieser Arbeit konnte gezeigt werden, dass an der TGF-beta 2-Signalgebung in humanen Trabekelmaschenwerkszellen außer dem klassischen Smad-Weg auch MEK/ERK und PI3K/AKT an der Signalübertragung beteiligt sind. Hierbei sind die TGF-beta-induzierten Änderungen der Zell-Zell-Verbindungen von der Smad- und AKT-Signalgebung abhängig, während die Effekte von TGF-beta auf alpha-SMA über den MEK/ERK-Signalweg vermittelt werden. Die Ergebnisse der vorliegenden Arbeit zeigen einige neu beobachtete TGF-beta-Effekte im Trabekelmaschenwerk, von denen insbesondere die Veränderungen des Zellskeletts und der Zell-Zell-Verbindungen sowie die möglicherweise stattfindende Depletion des Wnt-Signalweges Bedeutung für die Entstehung des Offenwinkelglaukoms haben könnten. / Primary open angle glaucoma (POAG) is a chronic optic neuropathy with elevated intraocular pressure and ageing as major risk factors. The incidence of POAG is expected to rise in the aging industrial societies. Structural changes in the trabecular meshwork (TM) and elevated TGF-beta 2 levels in the aequous humor have been described in POAG-patients. It has been shown that TGF-beta modulates the amount and the composition of the extracellular matrix and intracellular proteins in the TM. Here we show that TGF-beta 2 modulates the cytosceletal rearrangements and the expression of cadherins in human TM cells. These changes require the canonical Smad-pathway as well as non-canonical signaling by the Pi3K/AKT- and MEK/ERK-pathways. Changes in the cytoskeleton and cell-cell adhesions might influence the mechanotransduction characteristics of TM tissue and thus have effects on the regulation of intraocular pressure with implications in primary open angle glaucoma.

No description available.

No description available.

In unserem Institut wurde in vorangegangenen Studien der Nachweis für die Präsenz des Natrium-Glukose-Kotransporters (SGLT-1) im Pansenepithel von Schafen sowohl auf molekularbiologischer als auch auf funktioneller Ebene erbracht. Im Dünndarm von Monogastriern und Wiederkäuern hängt die Aktivität des SGLT-1 im Wesentlichen vom phylogenetischen Ernährungstyp (Wiederkäuer: Konzentrat-selektierer oder Rauhfutterkonsumenten), vom aktuellen Substratangebot und der Beeinflussung durch stoffwechselaktive Hormone ab. Es ist bekannt, dass bei vermehrter Aufnahme leicht verdaulicher Kohlenhydrate die Präsenz des intestinalen SGLT-1 erhöht ist. Eine Stimulation des SGLT-1 durch den intrazellulären Messenger cAMP ist beschrieben. Im Rattendünndarm wurde eine cAMP-vermittelte Erhöhung der Glukoseaufnahme durch pankreatisches Glukagon 29, enterisches Glukagon 37 und Adrenalin nachgewiesen. Die vorliegenden In-vitro- Studien am Pansenepithel waren der Frage gewidmet, inwiefern sich die bisher am Dünndarm untersuchten Regulationswege auf den ruminalen SGLT-1 übertragen lassen. Dies betraf im Einzelnen: - Phylogenetische Unterschiede bei der ruminalen SGLT-1-Aktivität zwischen verschiedenen Ernährungstypen (Schaf, Damwild) - Regulation des SGLT-1 durch kurzfristige Änderungen des Substratangebots - Steigerung der Glukoseaufnahme durch Hormone (Katecholamine u. Glukagon), Die Studien wurden mittels einer modifizierten Ussing-Kammer-Technik durchgeführt. Von Schafen und Damwild wurde die Tunica mucosa der Pansenepithelien präpariert und in Ussing-Kammern inkubiert. Nach einer einminütigen mukosalen Inkubation mit D-Glukose (einschließlich 14C-markierter Glukose) erfolgte die Bestimmung der Glukosekonzentration im Epithelzelllysat mittels Szintillationszählung der radioaktiv markierten Glukose. Der Einfluss verschiedener Substanzen auf die Glukoseaufnahme wurde durch entsprechende Vorinkubation untersucht. Es konnten folgende Befunde erhoben werden: - Die ruminale Glukoseaufnahme bei einer mukosalen Glukosekonzentration von 200 µM erfolgt bei Schafen zu ca. 60 % über den SGLT-1, bei Damwild betrug dieser Anteil nur etwa 25 %. - Eine mehrstündige Erhöhung des mukosalen Glukoseangebotes auf 10 mM resultierte in einer ca. 50%igen Steigerung der SGLT-1-vermittelten Glukoseaufnahme beim Schaf. - Die Hormone Glukagon 37 und Adrenalin bewirkten am Schafspansen eine deutliche Steigerung der SGLT-1-vermittelten Glukoseaufnahme. Glukagon 29 hatte einen geringen Effekt, während Noradrenalin die Glukoseaufnahme nicht nachweisbar beeinflusste. - Im Rahmen einer detaillierteren Charakterisierung des Adrenalineffektes konnte eine Stimulation des SGLT-1 auch durch den -Rezeptoragonist Isoproterenol und den 2-spezifischen Agonisten Terbutalin ausgelöst werden. Andere rezeptorspezifische Agonisten (alpha-1, alpha-2, beta-1 und beta-3) hatten keinen Einfluss. Schlussfolgernd lässt sich postulieren, dass der SGLT-1 bei Schafen der bedeutendste Transportmechanismus für die ruminale Glukoseaufnahme ist und bei Damwild im Gegensatz zu den vom Dünndarm bekannten Verhältnissen eine untergeordnete Rolle spielt. Eine Steuerung ist ähnlich wie im Dünndarm sowohl durch erhöhtes Glukoseangebot als auch durch Erhöhung der intrazellulären cAMP-Konzentration möglich. Die Hormone Adrenalin und Glukagon 37 stimulieren die Glukoseaufnahme in den getesteten Konzentrationen signifikant. Der Signalweg der Adrenalin-vermittelten Stimulation verläuft über Aktivierung der Adenylatzyklase und Proteinkinase A. Dafür scheint ein beta-2-Rezeptor im Pansenepithel verantwortlich zu sein. Die praktische Relevanz der untersuchten Stimulierbarkeit des SGLT-1 könnte in vivo in der schnellen Entfernung überschüssiger Glukosemengen aus dem Vormagen liegen, um eventuell einer bakteriellen Dysfermentation im Pansen entgegenzusteuern. / Recent studies evidenced the presence of the sodium / glucose cotransporter, SGLT-1, in ruminal epithelia of sheep and showed its ability to absorb glucose. Concerning the possibility of regulation of SGLT-1, the influence of substrate concentration has been reported in other tissues. There are phylogenetic adaptions according to the feeding habits of different ruminants (grass and roughage consumer, intermediate mixed feeder and concentrate selectors) and the possibility to short-term adaption in response to the availability of easely fermentable carbohydrates. Furthermore, some hormones are known to stimulate glucose uptake. The important role of cyclic AMP (cAMP) as an intracellular mediator and stimulator of glucose uptake is well described. There is evidence for the influence of hormones stimulating the SGLT-1 by rising the cAMP level. Stimulation of intestinal glucose uptake occured after preincubation with pancreatic glucagon (glucagon 29), enteric glucagon (glucagon 37) and epinephrine in the rat small intestine. To proove whether the regulatory priciples of the intestinal SGLT-1 apply also to the SGLT-1 in the ruminant forestomach, the heredescribed studies addressed the following topics: -Quantification of SGLT-1-mediated glucose uptake in the rumen of ruminants with different feeding habits (sheep vs. fallow deer) -Short-term regulation of glucose uptake by substrate availability -Modulation of glucose uptake by hormones with stimulatory effects on cAMP level Glucose uptake studies were performed in vitro using a modified Ussing chamber technique. Ruminal epithelia were incubated on the mucosal side with glucose (including 50 kBq 14C-glucose) for 1 min. Glucose content of the lysed epithelia was measured by scintillation counting. The influence of certain substances on glucose uptake was investigated by preincubation with these substances either on the serosal, mucosal or both sides of the epithelia. RESULTS - Ruminal glucose absorption in sheep is mediated to approximately 60 % by SGLT-1, whereas the SGLT-1 of fallow deer transports only 25 % of the glucose (at a mucosal glucose concentration of 200 µM). - After mucosal preincubation with 10 mM glucose for several hours, the SGLT-1-mediated glucose uptake of sheep rumen raised up to 150 %. - The hormones glucagon 37 and epinephrine increased SGLT-1-mediated glucose transport in the sheep rumen. A minor stimulation was also observed after addition of glucagon 29, whereas norepinephrine had no effect on glucose uptake. - The effect of epinephrine was characterized in detail. It could be simulated by using the beta-receptoragonist isoproterenol as well as the beta-2-receptor agonist terbutaline. No effect was observed with alpha-1-, alpha-2-, beta-1- and beta-3-receptor agonists. These results lead to the following conclusions: SGLT-1 is the most important transport mechanism for ruminal glucose uptake in sheep. In contrast to the high SGLT-1-activity in the intestine of fallow deer, there is only a minor relevance in the rumen of this species. A fast significant stimulation of the ruminal SGLT-1 of sheep in vitro is possible by rising glucose supply and serosal addition of epinephrine and glucagon 37 in the tested concentrations. Glucagon 29 and norepinephrine had no effect on SGLT-1 under the used experimental conditions. The intracellular signalling of the epinephrine action involves the pathway via adenylate cyclase and protein kinase A. The investigated effect of epinephrine is mediated by beta-2-adrenoceptors. The priciples of regulation of the ruminal SGLT-1 are similar to the intestine. Perhaps, in vivo, up-regulation of glucose absorption by substrates and hormones is important for a fast removal of excess glucose from rumen to prevent dysfermentation.

No description available.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves : [128]-155). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 196-228).

Exercise Induced Bronchoconstriction (EIB) is common amongst elite athletes. Short-acting β2-agonists represent the first-line treatment of EIB, however; limited data currently exists examining the ergogenic and pharmacokinetic impact of chronic short-acting β2-agonist administration. Furthermore, the ergogenic impact of acute and chronic administration of short-acting β2-agonists in asthmatic individuals is unknown. Whilst the short-acting β2-agonist salbutamol is permitted in and out of competition due to a known pharmacokinetic response, no urinary threshold has been established for the use of the alternative short-acting β2-agonist terbutaline. The purpose of study 1 was to investigate the ergogenic potential of the WADA upper daily limit of 1600 μg·day-1 salbutamol every day for 6 weeks versus placebo, alongside combined resistance and endurance training. Findings highlighted improvements in; 1 repetition maximum (1RM) bench press (Baseline: 65.6 ± 5.4 kg vs. 64.3 ± 4.9 kg – 6 weeks: 70.3 ± 4.9 vs. 72.5 ± 5.4 kg) and leg press (Baseline: 250 ± 26.9 vs. 217.9 ± 19 kg – 6 weeks: 282.5 ± 22.5 vs. 282.8 ± 18.3 kg); vertical jump test (Baseline: 53.5 ± 4.1 vs. 50.4 ± 2.1 cm – 6 weeks: 55 ± 3.5 vs. 52.4 ± 1.7 cm); 3 km running time-trial performance (Baseline: 988.7 ± 68.7 vs. 1040.5 ± 66.3 s – 6 weeks: 947.5 ± 54.9 vs. 1004.3 ± 70.5 s); isokinetic dynamometry (Baseline: 196.1 ± 47.3 vs. 184.6 ± 35.0 n.m. – 6 weeks: 179.5 ± 48.9 vs. 195.2 ± 28.9 n.m.); and body composition (Baseline: 32.1 ± 13.9 vs. 34.9 ± 10.4 mm – 6 weeks: 32.4 ± 14.5 vs. 34.5 ± 10 mm) for both the salbutamol group and the placebo group, respectively, over the 6 week period, with no difference observed between groups, indicating long-term therapeutic use of salbutamol at the WADA upper daily limit has no ergogenic effect. Of note, one participant exceeded the urinary threshold, presenting with an adverse 3 | P a g e analytical finding (AAF) showing that the upper daily limit can lead to AAF’s in susceptible individuals. Athletes who respond poorly to salbutamol treatment are able to apply for the use of the short-acting β2-agonist terbutaline via a therapeutic use exemption (TUE) certificate. Urinary upper limits are unknown for terbutaline and as such it is prohibited at all times without the presentation of a TUE. The purpose of study 2 was to investigate the urinary excretion of terbutaline following single and repeated use of inhaled or oral terbutaline. The aim of the study was to establish a differential distinction between routes of administration which could assist the WADA with regard to anti-doping policy and procedure. Results demonstrated a significant difference in urine concentration of terbutaline between inhaled and oral administration for female Caucasian (670.1 ± 128.3 vs. 361.8 ± 43.8 ng·ml-1; P=0.019; 680.8 ± 91 vs. 369.9 ± 41.9 ng·ml-1; P=0.006), male Afro-Caribbean (343.18 ± 45 vs. 231.3 ± 32.95 ng·ml-1; P=0.044; 389.73 ± 67.4 vs. 212.4 ± 50.3 ng·ml-1; P=0.008) and male Asian (266.4 ± 23.7 vs. 143.3 ± 22 ng·ml-1; P=0.004; 379.5 ± 50.4 vs. 197.5 ± 38.6 ng·ml-1; P=0.000) groups for single (5 mg oral vs. 2 mg inhaled) and repeated (4 x 5 mg oral vs. 8 x 1 mg inhaled) administration trials, respectively. No difference was observed in male Caucasians. High intra- and inter-individual variability between samples meant that a clear distinction between routes of administration could not be established. The study was able to identify an upper urinary threshold following inhaled administration of 1284.3 ng·ml-1 and an upper urinary threshold following oral use of 2376.3 ng·ml-1 which may inform the process of distinguishing between inhaled and oral use. Athletes are permitted to use inhaled terbutaline therapeutically through the TUE process. The purpose of study 3 was to investigate the ergogenic effect of terbutaline at high (2 mg and 4 mg) therapeutic inhaled doses on 3 km running time-trial performance in males and females. The 4 | P a g e study found that inhaled terbutaline, when used at the highest therapeutic dose, has no impact upon 3 km time-trial performance in males (956.3 s vs. 982 s) and females (1249 s vs. 1214.7 s) for placebo vs. 4 mg inhaled terbutaline, respectively. The majority of studies investigating the ergogenic potential of salbutamol have been in healthy individuals. It is not yet understood whether the exercise response differs in asthmatic individuals. The purpose of study 4 was to investigate the use of inhaled salbutamol (400 μg) during a 3 km running time-trial in eucapnic voluntary hyperpnoea positive (EVH+ve) and negative (EVH-ve) individuals, in a low humidity environment. Results demonstrated increased FEV1 in both groups following salbutamol inhalation, which did not translate to improved performance. No performance differences were found between salbutamol and placebo (Sal: 1012.7 ± 50 vs. 962.1 ± 37.5 s – Pla: 1002.4 ± 46.5 vs. 962 ± 28.8 s) in the EVH+ve group vs. the EVH-ve group, respectively. This thesis is the first to investigate the effects of long-term use of salbutamol at the WADA upper daily limit on exercise performance. It is also the first study to establish upper urinary thresholds for terbutaline use, and the effects of therapeutic inhaled terbutaline on exercise performance. The effect of salbutamol on exercise performance at low humidity in asthmatic individuals has also never previously been investigated. Overall, the findings from this thesis support previous research that inhaled β2-agonist use does not provide any ergogenic potential. With β2-agonists being an essential therapy for the treatment of EIB their current position on the WADA List of Prohibited Substances and Methods is appropriate. Further research is warranted to fully elucidate the upper urinary threshold for terbutaline to inform WADA and support the re-introduction of terbutaline as a therapeutic tool in the treatment of EIB in athletes.

Congenital myasthenic syndromes (CMS) are a rare group of heterogeneous disorders, characterised by compromised neuromuscular transmission and symptoms of fatiguable muscle weakness. CMS is caused by mutations in genes that affect the structure and function of the neuromuscular junction (NMJ). In about 20% of CMS cases, patients have mutations in the gene DOK7; the protein product, DOK7, is crucial for maintaining the dense aggregation of acetylcholine receptor (AChR) clusters at the NMJ. DOK7-CMS patients do not respond to treatment with acetylcholinesterase inhibitors which are the first line treatment for most forms of CMS. Instead, a dramatic response to beta-2 adrenergic receptor (ADRB2) agonists, such as salbutamol, is observed. The aim of this project was to investigate the molecular mechanisms that underlie the beneficial effects of ADRB2 agonists. Firstly, NMJ functioning was modelled in vitro by studying AChR clusters formed on cultured C2C12 mouse myotubes in the presence of WT DOK7. Overexpression of mutant DOK7 led to a significant reduction in the number of AChR clusters, explaining the pathogenic effect of the mutation. Importantly, incubation of myotubes with salbutamol increased the number of AChR clusters and their stability. The results provide the first evidence that ADRB2 agonists directly affect proteins located at the NMJ. However, this disease model suffers from limitations. The rest of the thesis focussed on developing alternative cell culture models to explore the AChR clustering pathway. The first model combined optogenetics and fluorescence lifetime microscopy to study the effects of ADRB2 activation on AChR cluster stability in single live cells. The second used CRISPR/Cas9 genome editing tools to directly introduce Dok7 mutations to the genome of C2C12 cells, thereby overcoming some of the drawbacks associated with DOK7 overexpression. Further manipulations of these novel model systems will be used in the future to examine in more detail the molecular events underlying the pathogenic effects of DOK7 mutations and the mechanisms of ADRB2 agonists.