Includes bibliographical references (leaves 73-79). / In vivo magnetic resonance spectroscopy (MRS) is an imaging technique that allows the chemical study of human tissue non-invasively. The method holds great promise as a diagnostic tool once its reliability has been established. Inter-scanner variability has, however, hampered this from happening as results cannot easily be compared if acquired on different scanners. In this study a phantom was constructed to determine the localisation efficiency of the 3 T Siemens Allegra MRI scanner located at the Cape Universities Brain Imaging Centre (CUBIC). Sufficient localisation is the key to acquiring useful spectroscopic data as only the signal from a small volume of interest (VOI) is typically acquired. The phantom consisted of a Perspex cube located inside a larger Perspex sphere. Solutions of the cerebral metabolites N-acetyl aspartate (NAA) and choline (Cho) were placed in the inner cube and outer sphere respectively. The phantom was scanned at a range of voxel sizes and echo times in order to determine parameters that typically indicate the performance of the scanner in question. The resultant full width at half maximum (FWHM) and signal to noise ratio (SNR) values indicated that optimal results were obtained for a voxel with dimensions 20 x 20 x 20 mm3. The selection efficiency could not be measured due to limitations in the scanner, but two other performance parameters ' extra volume suppression (EVS) and contamination ' could be determined. The EVS showed that the scanner was able to eliminate the entire background signal from the out-of-voxel region when voxel sizes with dimensions (20 mm)3 and (30 mm)3 were used. This performance decreased to 96.2% for a voxel size of (50 mm)3. The contamination indicated that the unwanted signal, weighted by the respective proton densities of the chemicals, ranged from 12% in the (20 mm)3 voxel to 24% in the (50 mm)3 voxel. These ranges are well within acceptable limits for proton MRS. Analysis of the water suppression achieved in the scanner showed an efficiency of 98.84%, which is acceptable for proton spectroscopy. It was also found that manual iv shimming of the scanner improved the spectra obtained, as compared to the automated shimming performed by the scanner. The second objective of the study was to quantify absolute metabolite concentrations in the familiar SI units of mM as results were previously mostly expressed as metabolite ratios. The LCModel software was used to assess two methods of determining absolute metabolite concentrations and the procedure using water scaling consistently showed superior performance to a method using a calibration factor. The method employing water scaling was then applied to a study of fetal alcohol spectrum disorder (FASD) where the deep cerebellar nuclei of children with FASD and a control group were scanned. The cerebellar nuclei were of interest as children with FASD show a remarkably consistent deficit in eye blink conditioning (EBC). The cerebellar deep nuclei is known to play a critical role in the EBC response. The results show significant decreases in the myo-inositol (mI) and total choline (tCho) concentrations of children with FASD in the deep cerebellar nuclei compared to control children. The FAS/PFAS subjects have a mean mI concentration of 4.6 mM as compared to a mean of 5.3 mM in the controls. A Pearson correlation showed that there was a significant relationship between decreasing mI concentrations with increasing prenatal alcohol exposure. The mean tCho concentrations are 1.3 mM for FAS/PFAS and 1.5 mM for the controls. There was no significant differences between the heavily exposed group and either the FAS/PFAS or the control subjects for either metabolite. The decreased mI and tCho concentrations may indicate deficient calcium signalling or decreased cell membrane integrity ' both of which can explain the compromised cerebellar learning in FASD subjects.

No description available.

Understanding how plants respond to stress is of importance, considering the increasing need to feed a growing population and supply its energy. Plants have complex systems for detecting, and responding to stresses. One stress-responsive system involves myo-inositol (Ins). Ins is a precursor for cell wall components, inositol trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol phosphate signaling molecules, and an alternate ascorbic acid (AsA) synthesis pathway. The enzyme, myo-inositol oxygenase (MIOX) is encoded by four genes in Arabidopsis and catalyzes the first step of Ins catabolism producing D-glucuronic acid (DGlcA). This research focuses on MIOX metabolism of Ins during plant growth and stress responses. I have examined miox mutants for alterations in metabolism and signaling. MIOX2 and MIOX4 expression patterns correlate with miox mutant root growth in varying nutrient conditions, and changes in flowering time. In miox2 mutants, I found an increase in Ins in most tissues, which was accompanied by cold- and abscisic (ABA)- sensitivity; however, miox4 mutants are ABA- insensitive, and have a small increase of Ins in flowers. MIOX2:GFP fusion protein accumulates in the cytoplasm and MIOX4:GFP accumulates in the cytoplasm and nucleus. Overexpresser MIOX4+ plants provide a model system to examine how directing carbon from Ins into DGlcA impacts Ins levels and Ins signaling. I have examined MIOX4+ plants for alterations in MIOX4 RNA and protein, and measured Ins by gas chromatography (GC). My results indicate that MIOX4+ tissues are impacted differently by the MIOX4 transgene, with decreases in Ins after seed imbibition, and increased Ins levels later in development. Ins depletion in seedlings was correlated with a decrease in Ins(1,4,5)P3. To determine the impact of reducing Ins and Ins(1,4,5)P3 in MIOX4+ seedlings, I examined processes known to involve Ins(1,4,5)P3 signaling. MIOX4+ seed have increased seed dormancy, NaCl-sensitivity, and ABA-insensitivity. These results suggest MIOX affects Ins signaling in response to ABA. Together, these data indicate that transcriptional control of MIOX2 and MIOX4 results in distinct roles in plant growth, and that MIOX2 and MIOX4 function in metabolic and signaling processes critical for growth, nutrient sensing, and stress responses. / Ph. D.

The objective of this research was to isolate genes encoding isoforms of the enzyme D-myo-inositol 3-phosphate synthase (MIPS, E.C. 5.5.1.4) from soybean and to characterize their expression, especially with respect to their involvement in phytic acid biosynthesis. A MIPS-homologous cDNA, designated GmMIPS1, was isolated via PCR using total RNA from developing seeds. Southern blot analysis and examination of MIPS-homologous soybean EST sequences suggested that GmMIPS1 is part of a multigene family of at least four similar members. The sequences of promoter and genomic regions of GmMIPS1 and GmMIPS2 revealed a high degree of sequence conservation. Northern and western blot analyses showed that MIPS transcript and protein are abundantly expressed early in seed development. Immunolocalization of MIPS protein in developing seeds confirmed expression of MIPS early in seed development and correlated MIPS protein accumulation in soybean seed tissue with tissues in which phytic acid is known to accumulate. The promoter region of GmMIPS1 was isolated and analyzed for possible seed-specificity using promoter:GUS fusions. Two GmMIPS1 promoter fragments were capable of conferring GUS expression when bombarded directly into developing soybean seeds. However, preliminary bombardment experiments into soybean cell suspension culture indicated that both promoter fragments drove expression of GUS in undifferentiated tissue, indicating a potential lack of seed-specificity. / Master of Science

Le myo-inositol (MI) est un soluté organique impliqué dans diverses fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2 est principalement localisé dans le cortex. Ces résultats ont été confirmés par immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous avons démontré que SMIT2 semble le seul responsable du transport luminal de MI dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de l’intestin, des études de transport de MI radioactif ont démontré une absence de transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est ii également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également testé la capacité de certains transporteurs de sucre présents à la surface des membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport. Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est incapable de transporter le MI. / Myo-inositol (MI) is an organic solute involved in various aspects of cell physiology, including cell signaling. It is also known as a compatible osmolyte. Three secondary active MI cotransporters have been identified; two are Na+- coupled (SMIT1 and SMIT2) and one is H+-coupled (HMIT). The main aim of this study was to characterize MI uptake throught SMIT2 as expressed in epithelial cells and in Xenopus laevis oocytes. In order to achieve the characterization of this transport system, we used purified brush border membrane vesicles (BBMv) isolated from rabbit kidney and rat intestine. We first performed a quantification of mRNA levels in rabbit kidney using real time PCR for both SMIT1 and SMIT2. We found that SMIT1 is mainly expressed in the renal medulla while SMIT2 is mainly localized in the renal cortex. This result was confirmed on Western blots using an antibody raised against SMIT2. Through inhibition studies using selective substrates for SMIT1 (inhibited by L-fucose) and SMIT2 (inhibited by D-chiroinositol), we showed that SMIT2 seems to be responsible for all the apical transport of MI into the proximal convoluted tubule with a Km of 57 ± 14 µM. By transport studies we established that rabbit intestine seems to lack apical transport of MI while rat intestine has a very active uptake of this molecule. qRT-PCR quantification confirmed the absence of MI transporters in rabbit intestine. As for kidney, SMIT2 seems to be the only transporter responsible for apical MI uptake in enterocytes with a Km of 150 ± 40 µM. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from rat intestine and rabbit kidney. SMIT2 displays high affinities for MI (270 ± 19 µM), DCI (310 ± 60 µM) and no affinity for L-fucose. SMIT2 is very sensitive to phlorizin (Pz; 16 ± 7 µM). Although these functional characteristics essentially confirmed those found in rat intestine, a iv discrepancy exists between the two systems studied. Indeed, the affinity constant for glucose was approximately 40-fold lower in vesicles than in oocytes. We also tested the ability of SGLT1 and GLUT5, other sugar transport systems present in enterocytes apical membranes, to perform MI uptake. Because the inhibition of these transporters did not alter radiolabeled MI uptake, we concluded that they had no significant contribution to MI transport in rat intestine. Finally, the basolateral efflux of MI was not mediated by GLUT2 because when expressed in oocytes, this transporter was not able to transport MI.

Le myo-inositol (MI) est un soluté organique impliqué dans diverses fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2 est principalement localisé dans le cortex. Ces résultats ont été confirmés par immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous avons démontré que SMIT2 semble le seul responsable du transport luminal de MI dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de l’intestin, des études de transport de MI radioactif ont démontré une absence de transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est ii également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également testé la capacité de certains transporteurs de sucre présents à la surface des membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport. Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est incapable de transporter le MI. / Myo-inositol (MI) is an organic solute involved in various aspects of cell physiology, including cell signaling. It is also known as a compatible osmolyte. Three secondary active MI cotransporters have been identified; two are Na+- coupled (SMIT1 and SMIT2) and one is H+-coupled (HMIT). The main aim of this study was to characterize MI uptake throught SMIT2 as expressed in epithelial cells and in Xenopus laevis oocytes. In order to achieve the characterization of this transport system, we used purified brush border membrane vesicles (BBMv) isolated from rabbit kidney and rat intestine. We first performed a quantification of mRNA levels in rabbit kidney using real time PCR for both SMIT1 and SMIT2. We found that SMIT1 is mainly expressed in the renal medulla while SMIT2 is mainly localized in the renal cortex. This result was confirmed on Western blots using an antibody raised against SMIT2. Through inhibition studies using selective substrates for SMIT1 (inhibited by L-fucose) and SMIT2 (inhibited by D-chiroinositol), we showed that SMIT2 seems to be responsible for all the apical transport of MI into the proximal convoluted tubule with a Km of 57 ± 14 µM. By transport studies we established that rabbit intestine seems to lack apical transport of MI while rat intestine has a very active uptake of this molecule. qRT-PCR quantification confirmed the absence of MI transporters in rabbit intestine. As for kidney, SMIT2 seems to be the only transporter responsible for apical MI uptake in enterocytes with a Km of 150 ± 40 µM. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from rat intestine and rabbit kidney. SMIT2 displays high affinities for MI (270 ± 19 µM), DCI (310 ± 60 µM) and no affinity for L-fucose. SMIT2 is very sensitive to phlorizin (Pz; 16 ± 7 µM). Although these functional characteristics essentially confirmed those found in rat intestine, a iv discrepancy exists between the two systems studied. Indeed, the affinity constant for glucose was approximately 40-fold lower in vesicles than in oocytes. We also tested the ability of SGLT1 and GLUT5, other sugar transport systems present in enterocytes apical membranes, to perform MI uptake. Because the inhibition of these transporters did not alter radiolabeled MI uptake, we concluded that they had no significant contribution to MI transport in rat intestine. Finally, the basolateral efflux of MI was not mediated by GLUT2 because when expressed in oocytes, this transporter was not able to transport MI.

Projektet skapades från viljan att framställa en virtuell träningsmiljö där blivande lärare kan öva och bättra på sin icke-verbala kommunikation när dem undervisar. Projektet funkar genom att fånga användarens rörelser och rendera virtuella agenter på en skärm framför användaren. Att stå framför en statisk skärm och undervisa kan dock inte kännas helt realistiskt. Denna rapport kommer gå igenom implementationen av en huvudmonterad display, mer specifikt Oculus Rift, för att skapa en virtuell verklighet och utökningen av de virtuella agenternas beetende och nya sätt att interagera med agenterna. De täcker även resultaten från experimenten där den nya funktionaliteten blev utvärderad. Experimenten gjordes genom att låta 18 personer testa systemet i både den gamla och nya uppsättningen och sedan fylla i frågeformulär efteråt. / This project was created from the desire to provide a virtual training environment for teachers-intraining to practice and improve their non-verbal communication with students. The project worked by capturing the user’s movements and rendering the virtual agent’s to a screen in front of the user. Standing in front of a static screen to hold a lecture might however not feel entirely realistic. This report covers the implementation of a head-mounted display, specifically the Oculus Rift, to create a virtual reality as well as the extension of the virtual agent’s behavior and new ways to interact with the virtual agent’s. It also covers the results of an experiment where the new functionality was evaluated. The experiment was done by allowing 18 persons to test the system in both the old and new configurations and fill in questionnaires afterwards.

Âncoras de glicosilfosfatidilinositol (GPI) são estruturas essenciais para a ancoragem de glicoconjugados e proteínas na superfície celular de protozoários. Trypanosoma cruzi produz uma gama de estruturas únicas de GPI, as quais ancoram mucinas e trans-sialidases, que participam de processos envolvidos na interação entre parasita e hospedeiro. Afim de estudar a biossíntese de âncora de GPI de T. cruzi e possivelmente utilizá-la como um potencial alvo anti-T.cruzi, este trabalho visa sintetizar análogos de âncoras de GPI e analisar o potencial destas moléculas como substratos da via biossintética de GPIs. Neste contexto, um pseudo-dissacarídeo 31 foi sintetizado através de O-glicosilação entre os doadores derivados de azido-glicopiranosídeo (32 ou 33a-d) e o acceptor de mio-inositol (34), preparados a partir de cloridrato de glucosamina (35) e metil-?-D-glucopiranósido (36), respectivamente, usando proteção/desproteção ortogonais. Cinco diferentes dadores de glicosídicos (32 e 33a-d) foram preparados para investigar a influcia dos seus grupos protetores na estereoselectividade da reações de O-glicosilação na presença de diferentes solventes para estudar o favorecimento da configuração ?, presente em GPIs. Ademais, a síntese do aceptor de mio-inositol 34 foi realizada em 12 etapas pela estratégia do rearranjo Ferrier para formar um derivado de ciclitol, além de diversas proteções/desproteções, funcionalizado que permite a introdução regiosselectiva da unidade de azido glicose (32-33a-d) e uma porção de fosfolípido no seu C-1 e posições C-6, respectivamente. Assim, O-glicosilação entre doador 33c e o acceptor 34, foi realizada utilizando TMSOTf como promotor para originar o composto 31c com boa estereoseletividade para ?, com elevado rendimento (~70%). Após a dealilação de 31c, a porção fosfodiéster contendo uma cadeia C-8 (87), preparada pela abordagem do H-fosfonato, foi anexada ao pseudo-dissacarídeo para gerar, após desprotecção global, o composto alvo 30a. A mesma estratégia sintética foi aplicada ao preparo do composto 91 contendo uma cadeia lateral alquil-naftil (90) que está em últmas etapas de desproteção para gerar o composto final 30c. Atualmente, o composto 30a está sendo testado como substrato da biossíntese de âncoras de GPI em membranas microssomais de Euglena gracilis, uma alga unicelular não patogênica, que pode potencialmente ser utilizada como modelo para parasitas humanos filogeneticamente relacionados. Após a incubação do potencial substrato de GPI 30a com membranas microssomais de E. gracilis para geração de metabólitos, será realizada análise do extrato por LC-MS e, eventualmente, isolamento dos produtos formados para posterior caracterização. Os produtos que apresentarem atividade como substrato ou como inibidores da biossíntese de GPI em E. gracilis serão também ensaiados na membrana microsomal do T. cruzi. / Glycosylphosphatidylinositol (GPI) anchors are essential molecules to attach glycoconjugates and proteins in protozoan\'s cell surface. Trypanosoma cruzi produces a range of unique GPI structures that anchor mucins and trans-sialidases which participate in important processes involved in the interaction between parasite and host. As an effort to study T. cruzi GPI anchor biosynthesis and possibly use it as a potential target for an antichagasic drug, this work aims to synthesize GPI anchor analogs (labelled or not) and analyze the potential of these molecules as substrates in the GPI biosynthetic pathway. In this context, a pseudo-disaccharide 31 was synthesized by O-glycosylation reaction between azide glycosyl donors (32 or 33a-d) and myo-inositol acceptor (34), prepared from glucosamine (35) hydrochloride and methyl ?-D-glucopyranoside (36), respectively, using orthogonal protection/ deprotection. Five different glycosyl donors (32 and 33a-d) were prepared to investigate the influence of their protective groups on the stereoselectivity of the O-glycosylation reaction in the presence of different solvents to afford the required GPI ?-linkage. In addition, the synthesis of the myo-inositol acceptor 34 was achieved using several protection/deprotection steps, besides the Ferrier rearrangement, to form a functionalized cyclitol derivative that enables the regioselective introduction of the azide glycoside unit and phospholipid moiety on its C-1 and C-6 positions, respectively. Then, O-glycosylation of acceptor 34 with donor 33c was accomplished in diethyl ether, using TMSOTf as promoter to give exclusively ?-anomer 31c in high yield. After deallylation of 31c, the phosphodiester moiety bearing an octyl chain (87), prepared by the H-phosphonate approach, was appended to the pseudo-disaccharide to yield, after deprotection, target compounds 30a. The same synthetic strategy was applied to the preparation of 30c, even though in the protective form, compound 91 bearing an alkyl-naphthyl side chain (90). Currently, compound 30a is being tested as substrates of GPI anchor biosynthesis in Euglena gracilis cell membranes, a non-pathogenic unicellular algae, which may potentially be used as a model for phylogenetically related human parasites. After incubation of the potential GPI substrate 30a with E. gracilis microsomal membranes for generation of metabolites, the analysis by LC-MS and, eventually, isolation of the products will be performed for further characterization. Products that show any substrate or inhibitory activities will be also assayed in T. cruzi microsomal membrane.

Parathormon (PTH) aktiviert am PTH-1 Rezeptor (P1R) mindestens zwei Signalwege: Über Guanosintriphosphat-bindende Proteine (Gs) kommt es intrazellulär zu einem Anstieg von zyklischem Adenosinmonophosphat (cAMP) und zu einer Aktivierung der Proteinkinase A (PKA). Gq Protein-vermittelt erfolgt eine Aktivierung der Phospholipase C (PLC) und ein intrazellulärer Anstieg des Inositoltriphosphat (IP3). Der verwandte PTH-2 Rezeptor (P2R) wird einerseits durch seinen vermutlich physiologischen Liganden, das tuberoinfundibuläre Peptid (TIP39), und andererseits durch PTH aktiviert. Im Gegensatz zum P1R zeigt der P2R nur eine Ankopplung an den cAMP Signalweg und keine an den PLC Signalweg. Voruntersuchungen hatten gezeigt, dass intrazelluläre Abschnitte der zweiten und dritten Schleife sowie Bereiche des C-Terminus des P1R für die Aktivierung des PLC Signalweges verantwortlich sind. In der vorliegenden Dissertation erfolgte eine stufenweise Angleichung intrazellulärer Abschnitte des P2R an den P1R. Die generierten Hybridrezeptoren wurden hinsichtlich ihres Signaltransduktionsverhaltens untersucht. Mit der Bestimmung der akkumulierten Gesamtinositole und des intrazellulären Calciums [Ca]i konnte gezeigt werden, dass trotz einer 95%-igen Übereinstimmung der intrazellulären Aminosäuresequenzen der P2R-Hybridrezeptoren mit denen des P1R, die Ankopplung an den PLC Signalweg nicht übertragen werden kann. Diese Ergebnisse wurden für Stimulationen mit PTH und TIP39 nachgewiesen. Durch Hemmung der Proteinkinasen A und C konnte, wie erwartet, am P1R ein verstärktes IP3 Signal beobachtet werden. Eine Aktivierbarkeit des PLC Signalweges wurde auch hierbei für die Hybridrezeptoren nicht gesehen. Für die Aktivierung des cAMP Signalweges der Hybridrezeptoren konnte beobachtet werden, dass diese sich weitestgehend in Anlehnung zum P2R verhalten. Außerdem konnte gezeigt werden, dass für eine effiziente Ankopplung an den cAMP Signalweg alle aus dem P1R in den P2R einfügten Teilabschnitte (zweite, dritte Schleife und C-Terminus) zusammenwirken. Der Hybridrezeptor, an dem alle drei Teilabschnitte ausgetauscht wurden, führte zu einer signifikant besseren Ankopplung an den cAMP Signalweg, als die Einzelmutation des C-Terminus. Die Messung zum cAMP Signalweg wurden mit den Liganden TIP39 und PTH durchgeführt. Für die Aktivierung des Gs Protein-vermittelten cAMP Signalweg am P1R oder P2R sind daher vermutlich Interaktionen verschiedener Rezeptorabschnitte verantwortlich. Insgesamt konnten in dieser Arbeit weitere wichtige Erkenntnisse bezüglich unterschiedlicher Aspekte der Signaltransduktion des P1R und des P2R gewonnen werden. / Activation of the phospholipase C (PLC) pathway upon stimulation with PTH is unique to the PTH-1 receptor (P1R) and is virtually absent in the PTH-2 receptor (P2R). The sites of P1R mutations known to reduce PLC signaling may achieve this indirectly by disturbing coupling at other sites. We therefore chose a more stringent approach and attempt to reestablish PLC signaling in the closely (70%) related P2R by gradually adapting the P2Rs cytosolic interface to a P1R-like sequence by engineering hybrid P1R/P2R constructs. Key differing regions of the P1R´s second loop (DT272-273:EK), third loop (IW344-345:LR), and C-terminal tail (T440-end: K-end) were put into the P2R. A fourth chimeric P1R/P2R receptor, combining all these changes, thus had a > 90% sequence identity with the cytosolic interface of the P1R. All mutants were expressed on the cell surface of stably transfected HEK 293 cells, using a radioreceptor assay with [125I]-Nle8,21-Tyr34-rat PTH(1-34), with a similar density of 1.1 to 2.5 mio receptors/cell. Activation of the PLC pathway was assessed by: 1) accumulation of total inositol phosphates in myo[3H]-inositol-prelabeled cells. No increase was detectable in the P2R or any of the receptor chimeras, even after enhancing the possible response by blockade of protein kinases A and C, despite an up to 13-fold increase with the P1R upon PTH stimulation. 2) Similarly, a PTH-induced increase in intracellular calcium (detected by fluo-3) of the wildtype P1R with as little as 1 nM hPTH(1-34) was completely absent in all mutants as well as in the P2R. The PTH-induced max. response of the cyclic AMP pathway was virtually identical for the P1R and P2R. However, the max. response of the mutant with the P1R tail sequence was reduced to only 35% (PTH) and 26% (TIP39) of the P2R response. Surprisingly, this signaling loss could be overcome by introducing more P1R sequence into the second and third cytoplasmic loop of this P1R/P2R hybrid receptor thus regaining a max. cAMP response of 64% (PTH) and 85% (TIP39). Presumably, an interaction of P1R sequences seems to result in a more effective coupling to the cAMP signaling pathway. In conclusion, these results suggest that the activation of the IP / intracellular calcium signaling pathway by the P1R is highly dependent on a P1R-like intracellular contact interface, which can not be mimicked easily even in the presence of > 90% of P1R sequence in the cytosolic interface of the P2R.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also called phytic acid, is ubiquitous in eukaryotic cells and the most abundant inositol phosphate derivative. Loblolly pine (LP, Pinus taeda) constitutes the primary commercial species in the southern forest of U.S. Somatic embryogenesis (SE) is an effective technique to maintain the desirable genetic composition of the progeny and to accomplish the efficiency of propagation. SE can also serve as a tool for study of plant development. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). In LP SE, FG tissue is absent in the embryogenic tissue culture. We found that extracts from early-stage FG stimulate growth and multiplication of early-stage somatic embryos, whereas FG water extracts from late stage contain substance(s) inhibitory to early-stage somatic embryo growth (DeSilva et al., 2007). We now present the isolation and identification of the inhibitory substance as InsP6 by means of water extraction, two gel filtrations and two ion exchange FPLC chromatographies. The results represent the first complete structural characterization of InsP6 from a natural product using LC/MS, LC/MS/MS, exact MS, 1D- and 2D-NMR analyses. We also report that there is a good correlation between the amount of InsP6 purified from FG tissue (1.3 nmoles per full-term FG) and the amount of InsP6 which inhibits somatic embryo growth. This novel approach of isolating and characterizing InsP6 from plant tissue, and investigating its role on SE can allow us to improve SE technology by circumventing current bottleneck, to elucidate enigmatic functions of InsP6 in plants, and most importantly, to utilize this molecule properly.