Structure-activity relationships for α-calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Conformational Evaluation of Indol-3-yl-N-alkyl-glyoxalylamides and Indol-3-yl-N,N-dialkyl-glyoxalylamides

Restricted rotation in indol-3-yl-N-alkyl- and indol-3-yl-N,N-dialkyl-glyoxalylamides can in principle give the syn-periplanar and anti-periplanar rotamers. In asymmetrically disubstituted glyoxalylamides, steric effects lead to the occurrence of both rotamers, as observed by NMR spectroscopy. The predominant peak corresponds with the anti rotamer, in which the bulkier alkyl group is orientated trans to the amide carbonyl group. In monoalkylated glyoxalylamides, only one set of peaks is observed, consistent with the presence of only one rotamer. Crystal structures of 5-methoxyindole-3-yl-N-tert-butylglyoxalylamide, indole-3-yl-N-tert-butylglyoxalylamide, and indole-3-yl-N-isopropylglyoxalylamide reported here reveal a syn conformation held by an intramolecular N-HO hydrogen bond.

Synthesis and characterization of silica-supported L-Lysine-based dendritic branches

Two series of novel modified silicas have been prepared in which individual dendritic branches have been attached to aminopropylsilica using standard peptide coupling methodology. The dendritic branches are composed of enantiomerically pure l-lysine building blocks, and hence, the modified silicas have the potential to act as chiral stationary phases in chromatography. In one series of modified silicas, the surface of the dendritic branch consists of Boc carbamate groups, whereas the other has benzoyl amide surface groups. Different coupling reagents have been investigated in order to maximize the loading onto the solid phase. The new supported dendritic materials have been fully characterized with properties of the bulk material determined by elemental analysis, 13C NMR, and IR spectroscopy, whereas XPS provides important information about the surface of the modified silica exposed to the incident X-rays, the key region in which potential chromatographic performance of these materials will take place. Although the bulk analyses indicate that loading of the dendritic branch onto silica decreases with increasing dendritic generation (and consequently steric bulk), XPS indicates that the optimum surface coverage is actually obtained at the second generation of dendritic growth.

New oil modified acrylic polymer for pH sensitive drug release:experimental results and statistical analysis

We report results of an experimental study, complemented by detailed statistical analysis of the experimental data, on the development of a more effective control method of drug delivery using a pH sensitive acrylic polymer. New copolymers based on acrylic acid and fatty acid are constructed from dodecyl castor oil and a tercopolymer based on methyl methacrylate, acrylic acid and acryl amide were prepared using this new approach. Water swelling characteristics of fatty acid, acrylic acid copolymer and tercopolymer respectively in acid and alkali solutions have been studied by a step-change method. The antibiotic drug cephalosporin and paracetamol have also been incorporated into the polymer blend through dissolution with the release of the antibiotic drug being evaluated in bacterial stain media and buffer solution. Our results show that the rate of release of paracetamol getss affected by the pH factor and also by the nature of polymer blend. Our experimental data have later been statistically analyzed to quantify the precise nature of polymer decay rates on the pH density of the relevant polymer solvents. The time evolution of the polymer decay rates indicate a marked transition from a linear to a strictly non-linear regime depending on the whether the chosen sample is a general copolymer (linear) or a tercopolymer (non-linear). Non-linear data extrapolation techniques have been used to make probabilistic predictions about the variation in weight percentages of retained polymers at all future times, thereby quantifying the degree of efficacy of the new method of drug delivery.

Synthesis and evaluation of N-(3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-1H-indole-carboxamides as cholecystokinin antagonists

The structure-activity relationship optimization of the pyrazoline template 3a resulted in novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides 4a-4e. These non-peptidal CCK ligands have been shown to act as potent CCK 1 ligands in a [125]I-CCK-8 receptor binding assay. The best amides (4c and 4d) of this series displayed an IC50 of 20/25 CCK 1 for the CCK 1 receptor. In a subsequent in-vivo evaluation using various behaviour pharmacological assays, an anxiolytic effect of these novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides was found at high doses in the elevated plus-maze. In the despair swimming test, a model for testing antidepressants, an ED50 of 0.33/0.41 mg kg -1 was determined for amide 4c/4d and the antidepressant effect had a magnitude comparable to desimipramine. © 2006 The Authors.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala8-substituted GLP-1 analogues

The hormone glucagon-like peptide-1(7-36)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucose-dependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the E-amino group in the side chain of the LyS26 residue and to combine this modification with substitutions of the Ala 8 residue, namely Val or amino-butyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal) 26]GLP-1, [Abu8,Lys(pal)26]GLP-1 and [Val8,Lys-(pal)26]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal β-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val8,Lys(pal)26]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)26]GLP-1, [Abu8,Lys(pal) 26]GLP-1 and [Val8,Lys(pal) 26]GLP-1 did not demonstrate acute glucose-lowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability. Copyright © by Walter de Gruyter.

Chemical characterization of dissolved organic nitrogen in an oligotrophic subtropical coastal ecosystem

Water samples were collected from rivers and estuarine environments within the Florida Coastal Everglades (FCE) ecosystem, USA, and ultrafiltered dissolved organic matter (UDOM; 1 kDa) was isolated for characterization of its source, bioavailability and diagenetic state. A combination of techniques, including 15N cross-polarization magic angle spinning nuclear magnetic resonance (15N CPMAS NMR) and X-ray photoelectron spectroscopy (XPS), were used to analyze the N components of UDOM. The concentrations and compositions of total hydrolysable amino acids (HAAs) were analyzed to estimate UDOM bioavailability and diagenetic state. Optical properties (UV–visible and fluorescence) and the stable isotope ratios of C and N were measured to assess the source and dynamics of UDOM. Spectroscopic analyses consistently showed that the major N species of UDOM are in amide form, but significant contributions of aromatic-N were also observed. XPS showed a very high pyridinic-N concentration in the FCE–UDOM (21.7 ± 2.7%) compared with those in other environments. The sources of this aromatic-N are unclear, but could include soot and charred materials from wild fires. Relatively high total HAA concentrations (4 ± 2% UDOC or 27 ± 4% UDON) are indicative of bioavailable components, and HAA compositions suggest FCE–UDOM has not undergone extensive diagenetic processing. These observations can be attributed to the low microbial activity and a continuous supply of fresh UDOM in this oligotrophic ecosystem. Marsh plants appear to be the dominant source of UDOM in freshwater regions of the FCE, whereas seagrasses and algae are the dominant sources of UDOM in Florida Bay. This study demonstrates the utility of a multi-technique and multi-proxy approach to advance our understanding of DON biogeochemistry.

O erro produzido pelo aluno no contexto pedagógico: uma luz ou uma pedra no meio do caminho?

Student’s mistakes as viewed in a didactic and pedagogical perspective are a phenomenon inevitably observed in any context in which formal teaching-andlearning processes are taking place. Researchers have shown that such mistakes are viewed most of the times as undesirable and often as a consequence of lack of attention or poor commitment on the part of the student and rarely considered didactically useful. The object of our reflections in this work is exactly those mistakes, which are born in the entrails of the teaching-and-learning processes. It is our understanding that a mistake constitutes a tool which mediates knowledge and may therefore become a strong ally of the instructor’s actions in her/his teaching tasks and thus should be taken into the teacher’s best consideration. Understanding a mistake as so, we postulate that the teacher must face it as a possibility to be exploited rather than as a negative occurrence. Such an attitude on the part of the teacher would undoubtedly render profitable didactic situations. To deepen the understanding of our aim, we took a case study on the perception of senior college students in the program of Mathematics at UFRN in the year 2009, 2nd term. The reason of this choice is the fact that Mathematics is the field presenting traditionally the poorest records in terms of school grades. In this work we put forth data associated to ENEM1 , to the UFRN Vestibular2 and the undergraduate courses on Mathematics. The theoretical matrixes supporting our reflections in this thesis follow the ideas proposed by Castorina (1988); Davis e Espósito (1990); Aquino (1997); Luckesi (2006); Cury (1994; 2008); Pinto (2000); Torre (2007). To carry out the study, we applied a semi-structured questionnaire containing 14 questions, out of which 10 were open questions. The questions were methodologically based on the Thematic Analysis – One of the techniques for Content Analysis schemed by Bardin (1977) – and it was also used the computer program Modalisa 6.0 (A software designed by faculties the University of Paris VIII). The results indicate that most of the teachers training instructors in their pedagogical practice view the mistakes made by their students only as a guide for grading and, in this procedure, the student is frequently labeled as guilty. Conclusive analyses, therefore, signal to the necessity of orienting the teachers training instructors in the sense of building a new theoretical contemplation of the students’ mistakes and their pedagogical potentialities and so making those professionals perceive the importance of such mistakes, since they reveal gaps in the process of learning and provide valuable avenues for the teaching procedures.

Micro-infiltration et rétention des scellants : évaluation de trois protocoles de mise en place

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Using Helix-coil Models to Study Protein Unfolded States

An abstract of a thesis devoted to using helix-coil models to study unfolded states.\\

Research on polypeptide unfolded states has received much more attention in the last decade or so than it has in the past. Unfolded states are thought to be implicated in various

misfolding diseases and likely play crucial roles in protein folding equilibria and folding rates. Structural characterization of unfolded states has proven to be

much more difficult than the now well established practice of determining the structures of folded proteins. This is largely because many core assumptions underlying

folded structure determination methods are invalid for unfolded states. This has led to a dearth of knowledge concerning the nature of unfolded state conformational

distributions. While many aspects of unfolded state structure are not well known, there does exist a significant body of work stretching back half a century that

has been focused on structural characterization of marginally stable polypeptide systems. This body of work represents an extensive collection of experimental

data and biophysical models associated with describing helix-coil equilibria in polypeptide systems. Much of the work on unfolded states in the last decade has not been devoted

specifically to the improvement of our understanding of helix-coil equilibria, which arguably is the most well characterized of the various conformational equilibria

that likely contribute to unfolded state conformational distributions. This thesis seeks to provide a deeper investigation of helix-coil equilibria using modern

statistical data analysis and biophysical modeling techniques. The studies contained within seek to provide deeper insights and new perspectives on what we presumably

know very well about protein unfolded states. \\

Chapter 1 gives an overview of recent and historical work on studying protein unfolded states. The study of helix-coil equilibria is placed in the context

of the general field of unfolded state research and the basics of helix-coil models are introduced.\\

Chapter 2 introduces the newest incarnation of a sophisticated helix-coil model. State of the art modern statistical techniques are employed to estimate the energies

of various physical interactions that serve to influence helix-coil equilibria. A new Bayesian model selection approach is utilized to test many long-standing

hypotheses concerning the physical nature of the helix-coil transition. Some assumptions made in previous models are shown to be invalid and the new model

exhibits greatly improved predictive performance relative to its predecessor. \\

Chapter 3 introduces a new statistical model that can be used to interpret amide exchange measurements. As amide exchange can serve as a probe for residue-specific

properties of helix-coil ensembles, the new model provides a novel and robust method to use these types of measurements to characterize helix-coil ensembles experimentally

and test the position-specific predictions of helix-coil models. The statistical model is shown to perform exceedingly better than the most commonly used

method for interpreting amide exchange data. The estimates of the model obtained from amide exchange measurements on an example helical peptide

also show a remarkable consistency with the predictions of the helix-coil model. \\

Chapter 4 involves a study of helix-coil ensembles through the enumeration of helix-coil configurations. Aside from providing new insights into helix-coil ensembles,

this chapter also introduces a new method by which helix-coil models can be extended to calculate new types of observables. Future work on this approach could potentially

allow helix-coil models to move into use domains that were previously inaccessible and reserved for other types of unfolded state models that were introduced in chapter 1.

Micro-infiltration et rétention des scellants : évaluation de trois protocoles de mise en place

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Design and Synthesis of 4-N-Alkanoyl and 4-N-Alkyl Gemcitabine Analogues Suitable for Positron Emission Tomography

Gemcitabine is a highly potent chemotherapeutic nucleoside agent used in the treatment of several cancers and solid tumors. However, it is therapeutically limitated because of toxicity to normal cells and its rapid intracellular deamination by cytidine deaminase into the inactive uracil derivative. Modification at the 4-(N) position of gemcitabine's exocyclic amine to an -amide functionality is a well reported prodrug strategy which has been that confers a resistance to intracellular deamination while also altering pharmacokinetics of the parent drug. Coupling of gemcitabine to carboxylic acids with varying terminal moieties afforded the 4-N-alkanoylgemcitabines whereas reaction of 4-N-tosylgemcitabine with the corresponding alkyl amines gave the 4-N-alkylgemcitabines. The 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues with a terminal hydroxyl group on the 4-N-alkanoyl or 4-N-alkyl chain were efficiently fluorinated either with diethylaminosulfur trifluoride or under conditions that are compatible with the synthetic protocols for 18F labeling, such as displacement of the corresponding mesylate with KF/Kryptofix 2.2.2. The 4-N-alkanoylgemcitabine analogues displayed potent cytostatic activities against murine and human tumor cell lines with 50% inhibitory concentration (IC50) values in the range of low nM, whereas cytotoxicity of the 4-N-alkylgemcitabine derivatives were in the low to modest µM range. The cytostatic activity of the 4-N-alkanoylgemcitabines was reduced by several orders of magnitude in the 2'-deoxycytidine kinase (dCK)-deficient CEM/dCK- cell line while the 4-N-alkylgemcitabines were only lowered by 2-5 times. None of the 4-N-modified gemcitabines were found to be substrates for cytosolic dCK, however all were found to inhibit DNA synthesis. As such, the 4-N-alkanoyl gemcitabine derivatives likely need to be converted to gemcitabine prior to achieving their significant cytostatic potential, whereas the 4-N-alkylgemcitabines reach their modest activity without "measurable" conversion to gemcitabine. Thus, the 4-N-alkylgemcitabines provide valuable insight on the metabolism of 4-N-modified gemcitabine prodrugs.

QUADRUPOLE CENTRAL TRANSITION NMR SPECTROSCOPY OF QUADRUPOLAR NUCLEI IN SOLUTION

This thesis reports on 17O (I = 5/2) and 59Co (I = 7/2) quadrupole central transition (QCT) NMR studies of three classes of biologically important molecules: glucose, nicotinamide and Vitamin B12 derivatives. Extensive QCT NMR experiments were performed over a wide range of molecular motion by changing solvent viscosity and temperature. 17O-labels were introduced at the 5- and 6-positions respectively: D-[5-17O]-glucose and D-[6-17O]-glucose following the literature method. QCT NMR greatly increased the molecular size limit obtained by ordinary solution NMR. It requires much lower temperatures to get the optimal spectral resolution, which are preferable for biological molecules. In addition, quadrupolar product parameter (PQ) and shielding anisotropy product parameter (PSA) were obtained for hydroxide group and amide group for the first time. For conventional NMR studies of quadrupolar nuclei, only PQ is accessible while QCT NMR obtained both PQ and PSA simultaneously. Our experiments also suggest the resolution of QCT NMR can be even better than that obtained by conventional NMR. We observed for the first time that the second-order quadrupolar interaction becomes a dominant relaxation mechanism under ultraslow motion. All these observations suggest that QCT NMR can become a standard technique for studying quadrupolar nuclei in solution.

Reversibly Photo-Crosslinkable Polycarbonate-Based Polymersomes for Drug Encapsulation and Delivery

This thesis describes the preparation of polymersomes from poly(ethylene glycol)-block-polycarbonate (PEG-PC) copolymers functionalized with pendant coumarin groups. Coumarin groups undergo photo-reversible dimerization when irradiated with specific ultraviolet wavelengths, so they can be used to prepare polymers with photo-responsive properties. In this case, the pendant coumarin groups enable stabilization of the polymersome membrane through photo-crosslinking of the hydrophobic block. Initially, several novel cinnamoyl and coumarin functionalized cyclic carbonate monomers were synthesized using ester, ether, or amide linkages. While the homopolymerization of these functionalized monomers proved challenging due to their high melting points, both cinnamoyl and coumarin functionalized monomers were successfully copolymerized with trimethylene carbonate (TMC) at 100 ℃ using a catalyst-free melt polymerization process where the TMC doubled as a solvent for the higher melting point monomer. Using this system, polycarbonate copolymers with up to 33% incorporation of the functionalized monomers were prepared. In addition, an investigation of some anomalous polymerization results identified previously unreported triethylamine-based catalysts for the melt polymerization of carbonate monomers. These studies also demonstrated that the catalyst-free polymerization of TMC occurs faster and at lower temperatures than previously reported. Subsequently, the photo-crosslinking of cinnamoyl and coumarin functionalized polycarbonates was compared and coumarin was identified as the more effective crosslinking agent when using 300-400 nm UV. An investigation of the photo-reversibility of the coumarin dimerization revealed no discernible change in the properties of crosslinked networks, but rapid photo-reversion in dilute solutions. The photo-crosslinking and photo-reversion kinetics of the coumarin functionalized polycarbonates were determined to be second-order in both cases. Finally, the self-assembly of PEG-PC diblock copolymers functionalized with coumarin was examined and both reverse solvent evaporation and solvent displacement were found to induce self-assembly, with hydrophilic mass fractions (f-factors) of 12-28% resulting in the formation of solid microparticles and nanoparticles and f-factors of 33-43% resulting in the formation of polymersomes. The stabilization of these polymersome membranes through photo-initiator-free photo-crosslinking was demonstrated with the crosslinking allowing polymersomes to withstand centrifugation at 12,000 x g. In addition, the encapsulation of calcein, as a model small molecule drug, in the stabilized polymersomes was successfully demonstrated using confocal microscopy.

Redox-dependent conformational switching of diphenylacetylenes

Herein we describe the design and synthesis of a redox-dependent single-molecule switch. Appending a ferrocene unit to a diphenylacetylene scaffold gives a redox-sensitive handle, which undergoes reversible one-electron oxidation, as demonstrated by cyclic voltammetry analysis. ¹H-NMR spectroscopy of the partially oxidized switch and control compounds suggests that oxidation to the ferrocenium cation induces a change in hydrogen bonding interactions that results in a conformational switch.