No description available.

Fibronectin (Fn) is an extracellular matrix (ECM) protein involved in embryonic development, wound healing and tumorigenesis. Structurally Fn is mainly composed of three repeated modules: FI, FII and FIII, together with an alternatively spliced type III connecting segment (IIICS). The IIICS has no sequence homology to these repeated modules and contains integrin, proteoglycan and zinc binding sites. These sites facilitate adherence and spreading of leukocytes, peripheral neurons and melanoma cells, which can lead to disease states such as inflammation, autoimmunity and cancer metastasis. Therefore, there is the potential to develop therapeutic agents based on the IIICS structure. In this study, nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the structure and dynamics of both the IIICS and its adjacent FIII15 module, two of the few Fn regions for which a structure has not been elucidated. An ensemble of solution state NMR structures calculated for the isolated FIII15 module showed that FIII15 forms a rare six-stranded FIII fold, homologous to a typical seven-stranded FIII fold, with a disordered N-terminal linker sequence. NMR relaxation data and chemical shift analysis showed that the IIICS is an intrinsically disordered region with no areas of well-defined secondary structure. A structure was also calculated for FIII15 within a construct containing the IIICS, which showed that contrary to a previous hypothesis, the IIICS does not contribute to the FIII15 structure. In addition, structural comparisons between IIICS splice variants suggested that alternative splicing confers no stable structural features to the IIICS. Furthermore, ligand binding studies showed that, under conditions tested, neither zinc nor the proteoglycan heparin, induced the formation of any secondary structure to this region. Zinc binding did, however, induce oligomerisation of a IIICS containing construct and appeared to enhance the binding of heparin to the IIICS. Data was obtained to suggest that FIII15 forms a transient interaction with an adjacent module, which is likely to be FIII14. It is hoped that the work presented will contribute to further studies into this important area of Fn and may aid in the future development of novel therapeutics.

No description available.

The rhomboid family of intramembrane serine proteases can catalyze proteolysis of substrates that are normally embedded in the cell membrane, making them key players in a diverse range of biological processes. While X-ray crystal structures provide detailed insights into the mechanism of intramembrane hydrolysis, questions remain concerning how transmembrane (TM) substrates are able to gain access to the rhomboid active site, and whether interactions with the membrane environment can influence its structure and function. In this thesis, these questions were investigated using the E. coli rhomboid ecGlpG. In Chapter 3, the effect of hydrophobic mismatch between lipid and protein was investigated using families of amphiphiles with saturated alkyl chains. While ecGlpG displayed maximal activity against a water-soluble model substrate when solubilized in detergents containing 10-12 carbon atoms, shorter and longer chain detergents led to loss of activity. An even larger effect was observed when ecGlpG was reconstituted into phospholipid bicelles, with no proteolytic activity being detected in 14-carbon lipids. These results suggest that mismatch between the hydrophobic regions of the catalytic TM domain (TMD) and the local membrane environment is detrimental to proteolysis. To obtain further insight into the structure and dynamics of ecGlpG, sample conditions were identified in Chapter 4 that enabled, for the first time, the acquisition of NMR spectra showing signals from the ecGlpG TMD. While significant peak broadening prevented chemical shift assignment, the sensitivity and resolution of peaks corresponding to the tryptophan indole NH group allowed their use as structural probes. These were employed in Chapter 5 to characterize the open conformation of ecGlpG that is postulated to facilitate substrate entry. These spectra showed evidence of an open conformation in which the intact α5 is laterally displaced. Interactions with a substrate-derived peptide also appeared to stimulate gate opening; however, activity assays suggested that formation of the open state could compromise catalytic activity against water-soluble substrates, and that interactions with TM substrates could counter this effect. Taken together, these results provide new insight into the role of both the local membrane environment and α5-conformational dynamics on intramembrane proteolysis, and suggest a mechanism to prevent cleavage of off-target rhomboid substrates in vivo.

No description available.

Cytoplasmic polyadenylation-element binding protein (CPEB) is required for the translational regulation in multiple cell types. CPEB is known to play important roles in early germ cell development, in neuronal synaptic plasticity, and in the process of cellular senescence. CPEB is able to control translation by first interacting with a specific sequence of mRNA known as the CPE site. CPEB recognizes a specific sequence of mRNA, called the cytoplasmic polyadenylation element. This is a uracil rich sequence that is located on the 3' UTR of mRNA. Once CPEB is bound to the CPE site, CPEB can interact with other proteins. CPEB is most notably known for interacting with a cleavage and polyadenylation specificity factor (CPSF), with a poly(A)-specific ribonuclease, and with a poly(A) polymerase in the Gld2 family. This complex of proteins controls polyadenylation on the 3' end of mRNA. By controlling the lengthening of the poly(A) tail, translation can be regulated. CPEB is believed to contain two RNA recognition motifs and a zinc binding region on the N-terminus. The zinc binding region contains six cysteine and two histidine residues that bind to two zinc atoms in a tetrahedral geometry. Using NMR spectroscopy, the structure of zinc binding region of CPEB1 was determined. This protein was shown to bind to two zinc ions in a cross-braced topology. The zinc binding region of CPEB was also determined that the correct classification for this zinc finger is a ZZ domain.

The ongoing effort to improve the sensitivity and information content of NMR spectroscopy and MRI has important implications in scientific research and medical diagnostics. In this dissertation, a variety of approaches have been investigated and expanded on in an effort to contribute to this field. First, cryptophanes are cage-shaped molecules that have previously been used to encapsulate molecules of interest for a number of potential applications--including gas sensing and biosensing. In one set of studies, encapsulation of molecular hydrogen gas (H2) has shown different behavior compared to other small organic molecules in C111 (up until now, the smallest cryptophane). The transient, non-covalent binding was studied by variable-temperature NMR at different fields up to 950 MHz. A mathematical model that considers multiple-H2 binding was developed to better understand the physics and binding process, with predictions compared to experimental data (and rationalized in light of quantum chemical calculations on possible H2@C111 complexes). To our knowledge, C111 is the only system to reversibly trap multiple H2 gas molecules non-covalently under mild conditions. In a second series of studies, the interaction of laser-polarized xenon and a water-soluble cryptophane was studied. Despite the low concentration of xenon in aqueous solution, it was possible to achieve polarization transfer from xenon to cryptophane spins via the SPINOE (spin-polarization induced nuclear Overhauser effect). The SPINOE enhancements, along with the 129Xe NMR spectra, provide information about the interaction of the Xe-cryptophane complex (variants of which are now used in so-called xenon biosensors). This was our first in-house successful application of hyperpolarized xenon as a signal source for the spins of other molecules, leading the way to a number of ongoing studies. Although the absolute NMR enhancements obtained via the SPINOE were small, much larger enhancements were studied in a technique that uses para-hydrogen (pH2)--a spin isomer of normal molecular hydrogen)--as the source of spin order. As with the xenon experiments (and the H2 binding experiments), pH2 must be delivered as a gas to a sealed sample prior to performing the NMR experiments. Parahydrogen-induced polarization (PHIP) is an emerging field in enhancing the sensitivity in NMR experiments and may play an important role in MRI studies. Within this field a very recent phenomena of signal amplification by reversible exchange (SABRE) was investigated. The reproducibility of this recent discovery has been examined and new conclusions about the mechanism of this technique are delineated. NMR signal enhancements of nearly ~400-fold are reported. Moreover, a new water soluble NHC-Iridium catalyst was synthesized and investigated in SABRE related studies. We also report the first studies of SABRE-enhancement in biologically tolerable solvents--opening a door to the development of SABRE-hyperpolarized metabolic contrast agents for subsecond molecular imaging in the body. Although much of the above work was motivated by the desire to improve NMR/MRI sensitivity enhancement, other efforts concerned the other side of the equation--improving NMR/MRI information content. The next section concerns our efforts to investigate use of Variable-Angle (VA) NMR to study composite liquid crystal (LC) media comprised of stretched polyacrylamide gels (SAG) and embedded bacteriophage Pf1 particles. This in situ combination exploited the apparent interference between the different solute-aligning properties of the two LC components--yielding composite media with alignment properties that can differ in a tunable manner from those obtained with each medium alone. Characterization of alignment of both large and small molecules provides more insight into the nature of solute alignment that those composite phases introduce--with the goal of developing this approach as a new technique for studying molecular structure and dynamics via the dipolar and quadrupolar couplings that are restored in liquid-crystalline media. Finally the use of SPIONS--superparamagnetic iron oxide nanoparticles--as contrast agents is a relatively new approach to enhance information content in MRI studies; this is particularly true for SPIONs that have been surface-functionalized to achieve an environment-sensitive MR response. Novel surface-functionalized SPIONs were investigated by examining their effect on nuclear spin relaxation in aqueous environments simulating bodily tissues. More specifically, the pH and ionic strength dependent properties of selected dendron-functionalized and polymer-functionalized SPIONs have been examined. Of particular interest to this dissertation is how environment-mediated transient clustering of the SPIONs gives rise to changes in so-called transverse (homogeneous) spin relaxation rates as measured by following the decay of MR signals detected after the application of a series of radio-frequency (RF) pulses. In order to better understand these effects in the context of the SPIONs' behavior, a mathematical model is under development whose predictions are compared with experimental data. Aspects of the model are also compared to transmission electron micrography (TEM) and dynamic light scattering (DLS).

Signal Amplification by Reversible Exchange, or SABRE, is a type of PHIP (ParaHydrogen Induced Polarization) pioneered by Duckett, Green, and co-workers where an organometallic catalyst is used to co-locate parahydrogen (pH2) and a molecular substrate to be hyperpolarized. Like traditional PHIP, SABRE is of interest because it is cost-effective, potentially continuous, scalable, and rapid (achieving polarization enhancement in seconds). However unlike traditional PHIP, SABRE does not require permanent alteration of the substrate to hyperpolarize it. In addition to achieving 1H polarizations of several percent, SABRE in microTesla fields has enabled the creation of ~10% polarization for heteronuclear (15N) spins. I will discuss on a series of novel catalysts that I developed in my Ph.D program. Firstly of all, a heterogeneous SABRE ("HET-SABRE") catalyst where catalytic moieties were tethered to solid supports. Although NMR enhancements were modest (5), this initial work showed the feasibility of the approach. Next, two types of nanoscale catalysts were created to explore SABRE at the interface between heterogeneous and homogeneous conditions. Nanoparticle and polymer comb variants were synthesized by covalently tethering Ir-based catalysts to support materials comprised of TiO2/PMAA (poly methacrylic acid) and PVP (polyvinyl pyridine), respectively, and characterized by AAS, NMR, and DLS. Following pH2 delivery to mixtures containing one type of "nano-SABRE" catalyst, a target substrate, and ethanol, up to ~(-)40-fold and ~(-)7-fold 1H NMR signal enhancements were observed for pyridine using the nanoparticle and polymer comb catalysts, respectively, following transfer to high field (9.4 T). These enhancements appear to result from intact particles and not from any catalyst molecules leaching from their supports. Unlike the case with homogeneous SABRE catalysts, high-field (in situ) SABRE effects were generally not observed with the nanoscale catalysts. The potential for separation and reuse of such catalyst particles is also demonstrated. Besides the effort on green chemistry of SABRE catalyst, I have been investigating the preparation of different variants of the "standard" SABRE catalyst--[IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD=cyclooctadiene)]--for performing SABRE in otherwise "pure" aqueous environments. Because of the poor aqueous solubility of SABRE catalysts, previous promising efforts have used co-solvents to achieve SABRE in aqueous/organic mixtures. However, I have found that the chemical changes that accompany this catalyst's activation also endow it with water solubility. Complete removal of the organic solvent following activation and subsequent re-constitution of the activated structure in deuterated water allowed up to ~(-)33-fold 1H signal enhancements to be obtained for nicotinamide. Additionally, I have investigated chemical alteration of the structure of the pre-activated catalyst to endow greater water solubility. PEGylation of the aromatic carbine moiety provided much greater aqueous solubility, but while SABRE-active in organic solutions, the catalyst lost activity in >50% water (an effect under ongoing study). As an alternative approach, synthesis of a di-Ir complex precursor where the COD rings have been replaced by CODDA (1,2-dihydroxy-3,7-cyclooctadiene) permits creation of a water-soluble catalyst [IrCl(CODDA)IMes] that enables aqueous SABRE in a single step without need for any organic co-solvent; the potential utility of the catalyst is demonstrated with the ~(-)32-fold enhancement of 1H signals of pyridine in water with only 1 atm of pH2. Taken together, these results support the utility of rational design for improving SABRE and HET-SABRE for applications varying from fundamental studies of catalysis to biomedical imaging. In the following, I also investigate different aspects of how catalyst structure can affect resulting SABRE enhancements, including the interplay of catalyst structure and temperature for optimal SABRE, as well as the confounding effects on catalyst activation. Results from the "standard" Ir SABRE catalyst (1)--[IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD=cyclooctadiene)]--are compared with those obtained with variants respectively created by synthetically replacing the -Cl moiety with 4-amino-pyridine (4AP, 2), (diphenylphosphino)ethylamine (DPPA, 3), triphenyl phosphine (TPP, 4), and tribenzyl phosphine (TBP, 5); a sixth variant (6) was serendipitously created by an alternate synthetic route for (1) that appears to result in a polymorph according to x-ray crystallography. Studies of activation rate found that (4) and (5) activated the fastest under pH2 exposure (~20 s, an order of magnitude faster than (1)); activation rate was inversely correlated with SABRE enhancement, with peak 1H polarization enhancement ( ranging from only ~(-)44 for (4) to nearly ~(-)1900 for (1) (or PH~6%) for pyridine at 9.4 T, and ~(-)240 for nicotinamide. Although (1) gave the overall highest  values as expected, other catalysts gave rise to better SABRE performance in other temperature regimes: Optimal temperatures varied significantly, e.g. ~273 K for (2) to ~310-320 K for (1); the optimal temperature for (6) was considerably lower (<273 K) than that for (1), despite the apparent structural similarity. Taken together, these results show that full optimization of SABRE enhancement for a given experiment (with respect to substrate, target nucleus, etc.) may require systematic variation of parameters including catalyst ligand choice and temperature (to modulate binding affinities and off rates with respect to relevant spin-spin couplings), in addition to pH2 partial pressure, flow rate, and magnetic field. Finally, some research on an ssNMR will be represented, to show the potential application of ssNMR on the coating detection.

The PsbQ protein (16.5 kDa) is an extrinsic protein found in the thylakoid membrane of higher plants and green algae. As a member of the Psb protein family, it is situated in the oxygen evolving center and takes part in the water splitting reaction. The stable oxygen production in photosystem II depends on the cooperation of PsbQ with other photosynthetic proteins, mainly PsbP. In order to identify the possible interaction sites, the tertiary structure in solution has to be determined. Although the X-ray crystallographic structure of PsbQ was determined previously, the conformation of residues 14-33 (so-called "missing link") was still unknown at the onset of this work. The initial backbone assignment as well as a secondary structure estimation were achieved recently. In this thesis the resonance assignment was extended and 15N as well as 13C NOESY-HSQC spectra were recorded to obtain structural constraints. The solution structure was determined using the program CYANA. The results obtained show that, while the four helix bundle domain is nearly identical compared to the available X-Ray crystallographic structure significant deviations occur in the N-terminal region. In particular, the residues 37-41, where a short ?-strand had been proposed in the crystal structure, exhibit high ?-helical propensity.

Made available in DSpace on 2016-08-29T15:35:36Z (GMT). No. of bitstreams: 1 tese_8107_LUCAS DE ALMEIDA GAMA20141202-143805.pdf: 4996710 bytes, checksum: ee8465315ab132ba2e49f39bf08cad52 (MD5) Previous issue date: 2014-08-27 / As análises de drogas de abuso são realizadas principalmente por técnicas analíticas como a cromatografia gasosa, podendo estar acoplada com a espectrometria de massas (CG/MS), e também técnicas de infravermelho (IV). Outras técnicas que antes eram inviáveis, tanto pelo alto custo dos equipamentos quanto pela baixa sensibilidade, agora estão se tornando técnicas viáveis para análises destas drogas. Um exemplo disso é a Ressonância Magnética Nuclear (RMN), que devido a necessidade de uma quantidade maior de amostra e ao alto custo de solventes deuterados, empregados na dissolução da amostra, tornavam a RMN uma técnica com alto custo agregado. Neste trabalho, foram realizados estudos de RMN sem a utilização de solvente deuterado na diluição das amostras de cocaína apreendidas pela Polícia Civil do Estado do Espírito Santo, denominada No-D NMR. A aplicação desta técnica diminui o custo das análises uma vez que não são utilizados compostos deuterados como solvente. Como se tratava de uma nova metodologia de análise, otimizou-se parâmetros de aquisição e de processamento característicos da RMN. A qualidade espectral obtida permitiu a distinção dos sinais característicos da cocaína e seus principais adulterantes: cafeína, fenacetina e lidocaína. Uma segunda etapa deste trabalho foi a aplicação de figuras de mérito para validar a No-D NMR como uma nova metodologia de quantificar a cocaína e seus principais adulterantes. Foram realizados estudos de especificidade/seletividade, linearidade, limite de detecção (LD), limite de quantificação (LQ) e exatidão/precisão que mostraram que a metodologia proposta é adequada para quantificar as substâncias em apreensões de cocaína. Após a obtenção das figuras de mérito, quantificou-se 29 apreensões que possuíam uma baixa quantidade de cocaína (cerca de 24% em massa) o que demonstrou que ocorre muita adulteração das amostras comercializadas no Espírito Santo.