Characterisation of bacterial multiple inositol polyphosphate phosphatases relevant to the animal feed industry

Phytases are enzymes that degrade phytate, the main storage form of phosphorus in plants. Animal feed industries use plant-based feed with abundant phytate content. Without the supplementation with phytases, the animals would not be able to access the phosphorus in the form of phosphate stored in the phytate molecule. This thesis describes research carried out to characterise a group of bacterial phytases from Bacteroides thetaiotaomicron, Bifidobacterium infantis and Bifidobacterium pseudocatenulatum with comparisons to an existing commercial phytase, Quantum Blue. The enzymatic properties, product profiles, binding and thermostability were examined and the structure of the binding site at various stages of the catalytic cycle was investigated with the aid of active site mutagenesis. X-ray structure of the active site mutant helped elucidate the structure of the intermediate and product-bound forms of the Bifidobacterium infantis phytase. Further mutagenesis experiments examined the function of disulphide bridges in the enzyme. The experimental results described in this work provide novel insights into the hydrolysis of phytate by bacterial phytases, the conformational changes during the catalytic cycle and the contribution of disulphide links to thermostability of the enzyme. These results lay the foundations of the work toward optimisation of phytases for use by the industry of the animal feed supplements

Etablierung der Peptid-Aptamertechnologie zur Modifikation des Carotinoid-Stoffwechsels in Pflanzen

In der vorliegenden Arbeit galt es, mithilfe der Peptid-Aptamer-Technologie ausgewählte Schlüsselenzyme des pflanzlichen Carotinoid-Stoffwechsels funktionell zu beeinflussen und so eine Anreicherung von Beta-Carotin zu erzielen. Als Zielenzyme wurden hierfür die drei plastidär lokalisierten Enzyme Phytoensynthase, Beta-Ring-Hydroxylase 2 und Lycopen-Epsilon-Cyclase aus dem Modellorganismus Arabidopsis thaliana ausgewählt. Nach Etablierung eines geeigneten bakteriellen Testsystems zur Carotinoid-Biosynthese konnten Funktion und Aktivität dieser Zielenzyme in E. coli eindeutig verifiziert werden. In Koexpressions-Experimenten kam es zu Zielprotein-abhängigen Verschiebungen der Carotinoid-Profile, und die entsprechenden Stoffwechselendprodukte wurden mittels Color-Complementation-Assay (Farbänderung der transgenen Bakterien) und HPLC-Analytik detektiert. Die Verwendung der funktionell charakterisierten Zielenzyme als Baits in Yeast-Two-Hybrid-Screenings führte zur erfolgreichen Identifikation spezifischer, interagierender Peptid-Aptamere aus verschiedenen Zufallspeptid-Libraries. Deren mögliche bioaktive Wirkung auf das jeweilige Zielenzym wurde in Koexpressions-Experimenten im bakteriellen Testsystem überprüft. Via HPLC-Analytik konnten so Aptamer-abhängige Veränderungen der Carotinoid-Metaboliten-Verhältnisse mit hoher Sensitivität detektiert und eindeutig nachgewiesen werden. Für das Zielenzym Lycopen-Epsilon-Cyclase (EC) wurde das 16 AS lange, bioaktive Aptamer 25Q6 mit stark inhibitorischer Wirkung identifiziert. Im Falle der Beta-Ring-Hydroxylase (BH2) wurde für 21 Aptamere ein vermutlich inhibierender Einfluss auf eine der beiden enzymatisch katalysierten Teilreaktionen nachgewiesen. Transiente Genexpressions- und BiFC (Bimolecular Fluorescence Complementation)-Studien in Nicotiana benthamiana bewiesen zweifelsfrei die funktionelle Expression und Interaktion von EC und 25Q6 in planta. Weitere Untersuchungen zur chloroplastidären Expression des 25Q6-Aptamers in transgenen Arabidopsis-Linien dienten der Bestimmung seiner bioaktiven Wirkung in Pflanzenzellen. Unter Verwendung des 35S-Promotors, des ChloroP-Transitpeptids und des Fluoreszenzmarkers CFP als Aptamer-Fusionsprotein konnte in transgenen Arabidopsis-Linien nur eine sehr schwache rekombinante Aptamer-Expression festgestellt werden. Die gewünschte chloroplastidäre Lokalisation konnte nicht nachgewiesen werden. Es wurden nicht aptamerspezifische, phänotypische Veränderungen der transgenen Pflanzenlinien festgestellt, die vermutlich durch CFP verursacht wurden. Zur funktionellen Charakterisierung der drei Zielgen-Promotoren wurden die vollständigen intergenischen upstream-Regionen transkriptionell mit dem GUS-Reportergen fusioniert und die Promotoraktivitäten über GUS-Expression in transgenen Arabidopsis-Linien hinsichtlich Stärke und Gewebe- bzw. Organspezifität analysiert. Alle drei Promotoren waren in A. thaliana aktiv, wobei sich die Expressionsstärken und -muster deutlich unterschieden. Eine verkürzte Variante des PSY-Promotors, bei der ein Teil des PSY-5'-UTRs und des ersten Introns fehlten, zeigte keine Reporter-Expression. Die Promotoren wurden desweiteren in transienten GUS-Expressionsanalysen in Früchten der Nutz- und Zielpflanze Tomate (Solanum lycopersicum) untersucht. Auch in dem heterologen System waren alle drei Promotoren aktiv und zeigten verschiedene Expressionsprofile. Der verkürzte PSY-Promotor war auch in den Tomatenfrüchten nicht aktiv

Hvordan kan norske bedrifter lykkes internasjonalt? En casestudie av internasjonaliseringsprosessen til innovative og nyetablerte bedrifter

Regjeringen har et mål om at norske bedrifter skal øke sin eksport til utlandet, men det er i den sammenheng viktig å også ta hensyn til fysisk etablering i utlandet. Det er fordi mange norske bedrifter velger en kombinasjon av eksport og direkte investering når de går internasjonalt. I denne masteroppgaven undersøker vi dermed internasjonaliseringsprosessen til norske innovative, nyetablerte bedrifter som etablerer seg i UK. Formålet med oppgaven er å finne ut hvordan bedriftene kan lykkes i utlandet med ulikt ressursgrunnlag og gjennom valg av ulike inngangsstrategier. Oppgaven er basert på en casestudie, hvor vi har gjennomført semi-strukturerte intervjuer med fire ansatte fra hver av de to casebedriftene: Saga Robotics og Plug. For å få bedre oversikt over markedet bedriftene har etablert seg i, har vi også intervjuet en rådgiver fra den britiske ambassaden. Studien baserer seg på teorier om internasjonaliseringsprosessen, inngangsstrategier og etableringsbarrierer. Det er gjort mye forskning på temaene, men flere av teoriene er ikke tilpasset det globale samfunnet vi har i dag. Vi vil dermed komme med et bidrag til teorien for å kunne se teoriene i lys av økt globalisering. Gjennom studien har vi avdekket at valget av inngangsstrategi baserer seg på hvilke ressurser bedriften sitter på, og egenskapene til bedriften. Funnene viser at ingen inngangsstrategi er bedre enn den andre, men heller at hver enkelt bedrift må identifisere sine ferdigheter og svakheter, og dermed kunne ta et fornuftig valg av inngangsstrategi. Studien viser også viktigheten av nettverket rundt bedriften. Det viser seg at nettverket er såpass viktig, at det virker å være umulig å internasjonalisere seg uten å ha en relasjon med noen i målmarkedet. Begge bedriftene har innovative produkter, og har dermed få konkurrenter. De har møtt på relativt lave etableringsbarrierer, men utfordringen har vært at de selv må bane vei i markedet. Gjennom korrekt valg av inngangsstrategi og gode nettverk, kan man få tilgang til de ressursene man mangler, og man er da bedre rustet til å håndtere etableringsbarrierene. Bedriftene opplever et konkurransefortrinn i dag, men siden markedet er umodent og under utvikling, gjenstår det å se om Plug og Saga Robotics beholder denne posisjon på sikt.nhhma

The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing.

Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion

Damage imaging in composites using nonlinear vibro‐acoustic wave modulations

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Morphological estimation of incomplete uterine scar rupture (dehiscence) in post- cesarean deliveries. Immunohistochemical studies

Objectives: No studies were found that analysed the properties of the caesarean scar, therefore the new study analysedthe myometrial immunohistochemical expression of elastin, collagen type VI, alpha smooth muscle actin, smooth musclemyosin heavy chain, and endothelial cell marker CD31.The aim of the study was to determine the risk of uterine rupture in future pregnancies.Material and methods: A total of 89 women of Caucasian ethnicity were eligible: 20 healthy pregnant women, who underwentrepeat caesarean section complicated by incomplete uterine scar rupture before labour, and 69 healthy pregnantwomen, who underwent repeat caesarean section without subsequent uterine scar rupture as the control group. In all cases,uterine tissue sample from the scarred region was collected during the caesarean section operation.Results: The lack of observed significant changes of elastin, collagen type VI, alpha smooth muscle actin, smooth musclemyosin heavy chain and endothelial cell marker CD31 concentrations in ruptured and unruptured uteri indicates that thesecomponents cannot be found to be a marker of risk of uterine rupture in future pregnancies.Conclusions: It could be suggested that the examined components do not contribute to the mechanism of maintainingintegrity and are not responsible for the biomechanical properties of the uterine scar