The regulation of metabolic and mitogenic cellular processes is a complex system, depending on the precise function of several signalling cascades. One example is the insulin pathway, which is mediated by a group of three sequentially and structurally highly similar hormones (insulin, insulin-like growth factor (IGF) -I and -II) and their homologous tyrosine kinase receptors (IR-A, IR-B, IGF-1R). Such a high degree of homology leads to crosstalk in receptor communication, with each of the ligands triggering different biological responses. The design of insulin analogues activating primarily metabolic effects or IGF antagonists suppressing an unfavourable mitogenic response is one of the main goals in this research field, which can be facilitated by identification of the regions responsible for receptor binding. The work included in this thesis is focused on the effects on receptor interactions following the introduction of selected elements from the IGF-I primary sequence into the IGF-II molecule. In particular, these include a point mutation of Ser29 to Asn, an insertion of Gly-Ser after Arg34 , an insertion of Pro-Gln after Ser39 , or combination of both insertions. Although the IGF-II modifications described here negatively affected binding affinity towards IR-A, they did not enhance the IGF-1R...

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Signal transduction kinases lie at the heart of the cell's ability to respond to environmental cues. These kinases are typically controlled by post-translational modification, most commonly by phosphorylation. S6K1alphaII is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (T389) regions of its catalytic kinase domain. In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID, residues 399-502), which inhibits T-loop and hydrophobic motif phosphorylation. Autoinhibition is relieved upon multi-site Ser-Thr phosphorylation of the AID by MAP kinase(s). We developed an optimized PCR-based gene synthesis method, which I utilized to build expression constructs for the AID alone as well as the kinase domain and full length S6K1alphaII. A fully activated form of S6K1alphaII was purified from Sf9 cells by co-expression with PDK1, and was used for in vitro analysis of the signaling pathway. AID was successfully purified in a soluble form from E. coli despite the fact that PONDR analysis predicted a highly disordered structure. Aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC spectra and far UV CD data showing a lack of secondary structure, confirmed that purified recombinant AID is largely unfolded. Despite this, addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1 and inhibition was decreased when the tetraphospho-mimic mutant AID(D2ED) was used. These studies, along with the reagents produced by them, will allow for further exploration of the emerging field of disordered regulatory domains.

A general picture of dendrimer conformation has appeared through studies of various dendrimer systems. Though the studies define some conformational abilities of a dendrimer, the studies are only able to examine one portion of the general picture. NMR studies of a generation three melamine dendrimer with unique NMR signatures from core to periphery describes most, if not all, of the general concepts of dendrimers in one system. A generation three melamine dendrimer was synthesized by a convergent route using diamines identified from competition reactions towards a monochlorotriazine. The cyclic monoamines surveyed displayed a relative reactivity range of 40x, expanding the previously identified series to a range of 320x. Azetidine is 40x more reactive than the cyclic, nine-membered ring (C8H17N), and 320x more reactive than benzyl amine. Sterics and pKa values explain the differences in reactivity of the cyclic monoamines. Differences in the nucleophilicity of the amine groups consisting of 2-aminoazetidine, 2-aminopyrrolidine, and 4-aminopiperidine are 180x, 70x and 20x, respectively. One-dimensional NMR spectra of the exchangeable NH region show that the dendrimer supports a rich rotamer population. Observations of the data show that the rotamer populations change from a preferred extended conformation to a more closed conformation, indicative of sterics being a driving force of conformational architecture. Variable temperature NOESY studies show that the peripheral groups backfold into the interior of the dendrimer in DMSO- d6. The backfolding can be removed by changing the solvent to either CDCl3 or CD3OD. Variable temperature (VT) coefficients measured for the exchangeable NH protons implies that solvent may be excluded from the interior of the dendrimer. Proton relaxation studies provide evidence that the dendrimer tumbles slowly in solution, and the periphery moves more freely than the interior. Synthesis towards the attachment of carboplatin-like peripheral groups on a generation three dendrimer was unsuccessful. A diethyl malonate unit was attached to the periphery of the dendrimer followed by capping with 4-aminomethylpiperidine. Hydrolysis of the esters and treatment with activated platinum led to a black precipitate product. Two alternate routes of achieving the desired platinated dendrimer are described.

A general picture of dendrimer conformation has appeared through studies of various dendrimer systems. Though the studies define some conformational abilities of a dendrimer, the studies are only able to examine one portion of the general picture. NMR studies of a generation three melamine dendrimer with unique NMR signatures from core to periphery describes most, if not all, of the general concepts of dendrimers in one system. A generation three melamine dendrimer was synthesized by a convergent route using diamines identified from competition reactions towards a monochlorotriazine. The cyclic monoamines surveyed displayed a relative reactivity range of 40x, expanding the previously identified series to a range of 320x. Azetidine is 40x more reactive than the cyclic, nine-membered ring (C8H17N), and 320x more reactive than benzyl amine. Sterics and pKa values explain the differences in reactivity of the cyclic monoamines. Differences in the nucleophilicity of the amine groups consisting of 2-aminoazetidine, 2-aminopyrrolidine, and 4-aminopiperidine are 180x, 70x and 20x, respectively. One-dimensional NMR spectra of the exchangeable NH region show that the dendrimer supports a rich rotamer population. Observations of the data show that the rotamer populations change from a preferred extended conformation to a more closed conformation, indicative of sterics being a driving force of conformational architecture. Variable temperature NOESY studies show that the peripheral groups backfold into the interior of the dendrimer in DMSO- d6. The backfolding can be removed by changing the solvent to either CDCl3 or CD3OD. Variable temperature (VT) coefficients measured for the exchangeable NH protons implies that solvent may be excluded from the interior of the dendrimer. Proton relaxation studies provide evidence that the dendrimer tumbles slowly in solution, and the periphery moves more freely than the interior. Synthesis towards the attachment of carboplatin-like peripheral groups on a generation three dendrimer was unsuccessful. A diethyl malonate unit was attached to the periphery of the dendrimer followed by capping with 4-aminomethylpiperidine. Hydrolysis of the esters and treatment with activated platinum led to a black precipitate product. Two alternate routes of achieving the desired platinated dendrimer are described.

The determination of reservoir quality and its spatial distribution is a key objective in reservoir characterization. This is especially challenging for carbonates because, due to the effects of diagenesis, quality rarely follows depositional patterns. This study integrates data from thin sections and core analyses with measurements of Nuclear Magnetic Resonance (NMR) T2 relaxation times. It exposes a novel approach to the use of NMR by applying geological and statistical analysis to define relationships between pore characteristics and the T2 data, from which a method to identify pore origin from NMR only is developed. One hundred and three samples taken from eleven wells located in fields of the Middle East, Alabama and Texas were used in the study. Modeling of the T2 spectra, as the sum of three normal components, resulted in the definition of 9 parameters representing the average, the variability and the percentage of total porosity of the specific pore sizes present in the sample. Each specific pore size corresponds to one of the following genetic pore types: intergranular, matrix, dissolution-enhanced, intercrystalline, vuggy and cement-reduced. Among the 9 parameters, two variables were identified as having the highest degree of geological significance that could be used to discriminate between pore categories: µmax which represents the largest average pore size of all pore types identified in the sample, and σmain which represents the size variability of the most abundant pore type. Based on the joint distribution of µmax and σmain computed for each pore category, the probability that an unclassified sample belongs to each of the pore categories, is calculated and the sample is assigned to the category with the highest probability. The accuracy of the method was investigated by comparing NMR predicted pore origin and genetic pore type described from thin section. A result of 89 successful predictions out of 103 samples was obtained. These promising results indicate that T2 time can be a useful identifier of carbonate pore types. Success in this work takes us closer to identifying genetic pore types from NMR logs with minimal calibration against borehole cores and will help predict the spatial distribution of poroperm facies in complex carbonate reservoirs with much improved accuracy.

The clathrates feature large cages of silicon, germanium, or tin, with guest atoms in the cage centers. The group IV clathrates are interesting because of their thermoelectric efficiency, and their glasslike thermal conductivity at low temperatures. Clathrates show a variety of properties, and the motion of cage center atoms is not well understood. In Sr8Ga16Ge30, we found that the slow atomic motion in the order 10-5 s is present in this system, which is much slower than what would be expected for standard atomic dynamics. NMR studies of Sr8Ga16Ge30 showed that Knight shift and T1 results are consistent with low density metallic behavior. The lineshapes exhibit changes consistent with motional narrowing at low temperatures, and this indicates unusually slow hopping rates. To further investigate this behavior, we made a series of measurements using the Carr-Purcell-Meiboom-Gill NMR sequence. Fitting the results to a hopping model yielded an activation energy of 4.6 K. We can understand all of our observations in terms of non-resonant atomic tunneling between asymmetric sites within the cages, in the presence of disorder. For Ba8Ga16Ge30, the relaxation behavior (T1) deviates from the Korringa relation, and the Knight shift and linewidth change with temperature. Those results could be explained by carrier freezout, and the development of a dilute set of magnetic moments due to these localized carriers. For Ba8Ga16Ge30 samples made from Ga flux, we observed different T1 and Knight shift behavior as compared to n type material. This is due to the differences in carrier type among these different samples. The p type sample has a smaller Knight shift and a slower relaxation rate than n type samples made with the stoichiometric ratio, which is consistent with a change in orbital symmetry between the conduction and valence bands. WDS study for Ba8Al10Ge36 showed the existence of vacancies in the Al-deficient samples, which results in some degree of ordering of Al occupation on the framework sites. In Al NMR measurements on Ba8AlxGe40-x with x = 12 to 16, we found that T1 of all Al samples follows the Korringa relation. The broadening of the single NMR central peak of Ba8Al16Ge30 is due to the inhomogeneous Knight shifts for occupation of different framework sites. For Ba8Al12Ge34 and Ba8Al13Ge33, we observed two peaks, and NMR results show that they are from distinct Al sites, while for each peak, the inhomogeneous broadening is much smaller. The difference in lineshapes we attributed to the existence of vacancies which we detected in the Al-deficient materials, and we assign one of the two Al peaks to Al adjacent to a vacancy.

Universiẗat, Diss.--Jena, 2007.

This thesis is concerned with the investigation of highly condensed, highly viscous silicate systems by means of NMR methods (mainly 29si-NMR investigations). The work focused on silicate solutions containing colloidal particles in sol and gel form with the interest of the work centred on the systems near the sol/gel-transition. Quantitative information about the behaviour of silicate species in these silicate systems is presented and some information about die role of protons and sodium cations in the silicate solutions is provided The structure of the colloidal material existing in these highly condensed silicate systems is investigated along with the behaviour of colloidal particles in several situations such as decreasing alkalinity or changing Si02-concentration. Additionally an idea of the range of particle sizes in the colloidal material in highly-condensed silicate solutions has been obtained. The dynamics of the silicate systems were studied in terms of rotational mobility as well as diffusive motion. Exchange processes between silicate species were monitored using ID-exchange spectroscopy. An investigation of the influence of additives, in particular surfactants, on highly condensed silicate systems was carried out by adding representative surfactants of the categories cationic, anionic and non-ionic.