677 research outputs found

    Relating starch structure in rice to its digestibility

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    Rice is a globally important crop and a major staple for over two-thirds of the world’s population. Australian grown rice is renowned for its high and consistent quality and is the preferred choice in the domestic and many international rice markets. A long-established focus on quality in the Australian rice breeding program has led to a range of new varieties for different cuisines, for example sushi, long grain, medium grain and fragrant rice types. Development of new varieties takes up to 10 years from parental cross to a pure seed line that boasts sound agronomic, pest and disease resistance and the desired combination of quality traits. Selection techniques vary for each generation, and for each trait. This is a huge undertaking with upwards of 6500 breeding lines assessed for physical quality each year, and more than 3000 samples assessed for cooking qualities. As new consumer trends emerge, new market opportunities for rice are uncovered such as the recent shift toward more health-conscious consumer decisions. Development of more varieties with lower glycaemic index is one avenue to explore further. For this, additional, more well-understood tools are required to measure, predict and/or actively select for this trait at different stages of the breeding and quality program. Apparent amylose content is currently the only published link available with which researchers can predict the digestibility characteristics of a given rice sample. While the correlation between these attributes is good (r2 = 0.73), it is also indicative that there are other drivers at play. This is highlighted in instances where glycaemic index can vary by up to 20 points at a given apparent amylose content. There is a gap in the understanding of which levels of starch structure, if any, can account for the differences observed in digestibility where apparent amylose content is similar. To explore this, multiple levels of starch structure were assessed in different rice varieties using a combination of novel and well-established characterisation methods. The determination of factors of starch structure relevant to digestibility in this study was intended to better understand the drivers of digestibility in rice grains. And through this, attempt to provide new tools with which to assess samples likely to exhibit a higher or lower digestibility, thus allowing for better selectivity in breeding where certain digestibility characteristics are a grain quality goal. This was achieved through the characterisation of multiple features of starch structure which were found to provide valuable input to refining the understanding of rice digestibility

    Experimental versus theoretical log D<sub>7.4</sub>, pK<sub>a</sub> and plasma protein binding values for benzodiazepines appearing as new psychoactive substances

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    The misuse of benzodiazepines as new psychoactive substances is an increasing problem around the world. Basic physicochemical and pharmacokinetic data is required on these substances in order to interpret and predict their effects upon humans. Experimental log D7.4, pKa and plasma protein binding values were determined for 11 benzodiazepines that have recently appeared as new psychoactive substances (3‐hydroxyphenazepam, 4’‐chlorodiazepam, desalkylflurazepam, deschloroetizolam, diclazepam, etizolam, flubromazepam, flubromazolam, meclonazepam, phenazepam and pyrazolam) and compared with values generated by various software packages (ACD/I‐lab, MarvinSketch, ADMET Predictor and PreADMET). ACD/I‐LAB returned the most accurate values for log D7.4 and plasma protein binding while ADMET Predictor returned the most accurate values for pKa. Large variations in predictive errors were observed between compounds. Experimental values are currently preferable and desirable as they may aid with the future ‘training’ of predictive models for these new psychoactive substances

    Novel Kinetic Solution-Based Separation Approaches for Small Molecule Drug Discovery

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    The modern pharmaceutical industry has achieved remarkable successes in medicinal chemistry. However, many diseases are incurable due to the difficulty of finding new drugs. De novo drug discovery contains two steps: the primary screening focuses on selecting protein (target) binding drug (ligand); the secondary screening concentrates on calculating kinetic binding parameters of target-ligand complex. Conventional methods for the primary screening are typically surface-based, which suffer intensely from nonspecific interactions; the existing methods for secondary screening are either affinity-based or require surface immobilization, both cannot accurately calculate kinetic binding parameters. Hence, this research focuses on the development of the solution-based kinetic platform that facilitates both primary and secondary screenings. We combined kinetic capillary electrophoresis (KCE) with DNA-encoded ligand (DEL) technology to build a solution-based platform for primary screening of ligands. KCE offers high partitioning efficiency but requires the knowledge of electrophoretic mobility of target-ligand complex, and thus, we developed a mathematical model to predict electrophoretic mobility of target-DEL complex. This model was tested by using the targets interacted with 18 artificial DELs that contain various combinations of dsDNA and ssDNA regions, together with 2 DELs manufactured by GlaxoSmithKline. The results confirmed the precision, accuracy, and ruggedness of our model. This model will facilitate the reliable use of KCE-DEL based primary screening. Next, we developed a kinetic size-exclusion chromatography-mass spectrometry (KSEC-MS) as the label-free solution-based platform for calculating kinetic binding parameters of target-ligand interactions in secondary screening. KSEC-MS employs size-exclusion chromatography to separate small molecule ligand from protein target-ligand complex without immobilization and mass spectrometry to detect small molecule without a label. The rate constants of complex formation and dissociation are calculated from the temporal ligand concentration profile. Methods of KSEC-MS have been developed by using 2 proteins with the corresponding drugs. The resulted kinetic and affinity binding parameters were validated, which confirmed the precision and accuracy of KSEC-MS. We foresee that the KSEC-MS will become a universal approach for the kinetic analysis of target-ligand interactions in secondary screening

    The characterisation of ultrafiltration membranes used in water purification

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    The increase of urbanisation has caused water scarcity concerns in developed nations. Natural sources of freshwater (ground water, river water, rain fall) are becoming insufficient, requiring man-made technologies for the purification of water. Ultrafiltration membranes are currently becoming more relied upon for water purification due to their selectivity and applicability in different environments. Fabrication of some ultrafiltration membranes require the mixing of two (binary blend) or three (ternary blend) synthetic polymers to form an amphiphilic polymer blend. Overall properties of the blend are dependent on the chemical nature of the precursors; however, chemical incompatibilities between polymers causes incomplete mixing, thereby forming a partially miscible system. The presence of different domains of varying miscibility creates a complex matrix where minimal changes in local chemical composition can drastically change the membranes properties. The material becomes increasingly complex with a ternary blend. Hence, the understanding of function and composition relationship is important to the development of design and functionality. This project aims to characterise a number of properties of ultrafiltration membranes at various stages, from fabrication to production. In this work, industrial membranes (both binary and ternary) and their respective precursors were studied. Difference in end groups within the system affects solubility of the precursor leading. This can lead to undissolved poly(N-vinyl pyrrolidone) (PVP) affecting the properties of the membrane produced. Free solution Capillary Electrophoresis (CE) was employed for the separation of PVP via end groups. Representative electrophoretic mobility distributions of different PVP samples were obtained showing the presence of different populations. The membranes were characterised through solid-state Nuclear Magnetic Resonance (NMR) spectroscopy, providing a means to determine the molecular structure and molecular mobility within the membranes. NMR measurements identified that polymer A is extracted out of the membrane during production and the ternary membrane is a miscible system. Deductions made contributed to interpretations on functionality Relationships between the surface composition and the functionality of the membrane were established. Functional properties were determined through tensile strength tests and tensiometry tests. The surface composition was determined through Scanning electron microscopy / energy dispersive x-ray spectroscopy and 1H NMR spectroscopy. Surface localisation of polymer A and polymer C affected the hydrophilicity of the membranes. It was also found the formation of macrovoids affect the tensile strength of the membrane. In conclusion, methods were developed to determine the chemical structures of the membrane, at various stages of production, and relating it to functionality. Analysis of structure-function relationships allowed for the improvement of design to optimise membrane properties. The AB membrane was determined to have better functional properties than the ABC membrane; however, the chemical stability of the ABC membrane makes it a promising system to be used for future design. Future design proposals can incorporate different polymerisations yielding PVP with end groups that are soluble. These improved designs will not only increase performance but also allow for cost-efficient measures of membrane production

    Electrokinetic Modeling of Free Solution Electrophoresis

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    Modeling electrophoresis of peptides, proteins, DNA, blood cells and colloids is based on classical electrokinetic theory. The coupled field equations-Poisson, Navier-Stokes or Brinkman, and ion transport equations are solved numerically to calculate the electrophoretic mobilities. First, free solution electrophoretic mobility expressions are derived for weakly charged rigid bead arrays. Variables include the number of beads (N), their size (radius), charge, distribution (configuration), salt type, and salt concentration. We apply these mobility expressions to rings, rigid rods, and wormlike chain models and then apply the approach to the electrophoretic mobilities and translational diffusion constants of weakly charged peptides. It is shown that our bead model can predict the electrophoretic mobilities accurately. In order to make the method applicable at higher salt concentrations and/or to models consisting of larger sized subunits, account is taken of the finite size of the beads making up the model structure. For highly charged particles, it is also necessary to account for ion relaxation. This ion relaxation effect is accounted for by correcting unrelaxed mobilities on the basis of model size and average electrostatic surface, or zeta potential. With these corrections our model can be applied to the system with absolute electrophoretic mobilities exceeding approximately 0.20 cm2/kV sec and also models involving larger subunits. This includes bead models of duplex DNA. Along somewhat different lines, we have investigated the electrophoresis of colloidal particles with an inner hard core and an outer diffusive layer ( hairy particles). An electrokinetic gel layer model of a spherical, highly charged colloid particle developed previously, is extended in several ways. The charge of the particle is assumed to arise from the deprotonation of acidic groups that are uniformly distributed over a portion (or all) of the gel layer. Free energy considerations coupled with Poisson-Boltzmann theory is used to calculate the change of the local pKa of the acidic groups depending on the local electrostatic environment. Based on the modeling of electrophoresis and viscosity, we predict that the thickness of the gel layer decreases as the salt concentration increases. And only the outermost portion of the gel layer is charged

    High throughput determination of relevant physicochemical parameters in the drug discovery and HPLC processes. Microfluidic devices

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    [eng] Determining the acidity (pKa) and lipophilicity (log Po/w) of organic compounds is fundamental in analytical chemistry fields, with potential relevance in drug development, material science, analytical separation, and environmental research. In the first part of the thesis, a high-throughput internal standard capillary electrophoresis (IS- CE) method was established to determine the pKa of ISs at different concentrations of methanol and acetonitrile from 0 to 90% (v/v). The acid and base scales of methanol-water mixtures and acetonitrile-water mixtures were properly anchored to the potentiometrically obtained pKa values of reference compounds to get absolute pKa scales. As a consequence, a set of 46 acid- base compounds with changing structures were proposed as internal standards for consistent pKa measurements in methanol-water and acetonitrile-water mixtures buffers using capillary electrophoresis. The determined ISs reference set facilitates the determination of analytes pKa and measurement of buffer pH in the range 4-11.5 (in water) for any methanol-water and acetonitrile-water composition. Secondly, to prove its feasibility, the IS-CE approach was successfully used to determine the aqueous pKa in methanol-aqueous buffer compositions up to 40% of methanol in volume. The Yasuda-Shedlovsky extrapolation method was utilized to determine seven drugs of different chemical nature with intrinsic water solubilities lower than 10−6 M. The results were successfully compared to literature ones obtained by other approaches. It is concluded then that the IS-CE methodolgy permits the measurement of aqueous pKa values using lower ratios of methanol than the classical method, becoming then more accurate in the extrapolation procedure than other reference methods. Finally, since methanol-water and acetonitrile-water mixtures are solvents of interest in liquid chromatographic separations because of their use as the mobile phase, the IS-CE method was also applied to measure the pKa of eight organic bases in methanol-water and acetonitrile-water mixtures (0-90%,v/v), which are usually used as test compounds in HPLC column evaluation. In the second part of the thesis, a new approach based on microfluidics was developed to determine the octanol-water partition. As a first step, a design with a perpendicular configuration of the channels was developed using direct 3D printed microfluidics. A gravitational perfusion system was implemented to create a spontaneous flow within the octanol and water channels without need for external pump. The movement of octanol and water phases was successfully validated using fluorescent dyes. After that, the intensity of the fluorescent dye was used to evaluate the partition dynamics in static and dynamic conditions. The results prove that the proposed design with this microfluidic methodology allows the evaluation of molecule partition, achieving high efficiency partition and reaching the equilibrium of O/W partition faster than conventional techniques. Later, the design was adapted to a parallel configuration of the channels to be compatible with up-scalable manufacturing techniques and parallelize it for up to 56 simultaneous determinations in a single platform. Finally, both the perpendicular and parallel designs were validated using several drugs with well standardize log Po/w values that cover a wide range of lipophilicity. The microfluidic device was coupled with HPLC to determine their partition coefficients from the peak areas of the compounds in octanol and in water after partition. Good agreement with the literature values was achieved, showing the capability of microfluidic chips for precise and accurate prediction of the partition coefficient. Finally, the progress of a cost-effective and consistent method for predicting partition coefficient via microfluidic chips demonstrated a great advancement in the field of analytical chemistry, with powerful applications in drug discovery and other related fields. The results gotten from this investigation offer an establishment for additional research and advance of this approach.[cat] La determinació de l'acidesa (pKa) i de la lipofilicitat (log Po/w) dels compostos orgànics és fonamental en els camps de la química analítica, amb rellevància potencial en el desenvolupament de fàrmacs, la ciència dels materials, la separació analítica i la investigació ambiental. En la primera part de la tesi s’estableix un mètode d'electroforesi capil·lar estàndard interna’'alt rendiment (IS-CE) per determinar el pKa de’'analit àcid-base en mescles d'aigua amb solvents orgànics. En aquest treball es va establir un mètode d'electroforesi capil·lar estàndard d'alt rendiment (IS-CE) per determinar el pKaa dels IS a diferents concentracions de metanol i acetonitril del 0 al 90% (v/v). En segon lloc, per demostrar la seva viabilitat, L'enfocament IS-CE es va utilitzar amb èxit per determinar el pKa aquós en composicions tampó aquoses de metanol fins a un 40% del volum de metanol. El mètode d'extrapolació De Yasuda-Shedlov es va utilitzar per determinar set fàrmacs de diferent naturalesa química amb solubilitats d'aigua intrínseques inferiors a 10-6 M. Aplicat el mètode IS-CE per mesurar el pKa de vuit bases orgàniques en mescles de metanol- aigua (0 -90%, v/v) i d’acetonitril-aigua, que s’utilitzen habitualment en l'avaluació de columnes de HPLC. A la segona part de la tesi, es va desenvolupar un nou mètode basat en la microfluídica per determinar la partició octanol-aigua. Com a primer pas, es va desenvolupar un disseny amb una configuració perpendicular dels canals mitjançant microfluídica impresa En 3d Directa. Es va implementar un sistema de perfusió per gravetat per crear flux espontani dins dels canals d'octanol i aigua sense necessitat de bombament extern. El moviment de les fases d'octanol i aigua es va validar amb èxit mitjançant colorants fluorescents. Després d'això, es va utilitzar la intensitat del colorant fluorescent per avaluar la dinàmica de partició en condicions estàtiques i dinàmiques. Posteriorment, el disseny es va adaptar a una configuració paral·lela dels canals per ser compatible amb tècniques de manufacturar up-scalable i parallelitzar-lo fins a 56 determinacions simultànies en una sola plataforma. Finalment, tant els dissenys perpendiculars com els paral·lels es van validar utilitzant diversos fàrmacs amb valors log Po/w de registre ben estandarditzats que cobreixen una àmplia gamma de lipofilicitat. El dispositiu microfluídic es va acoblar amb HPLC per determinar els seus coeficients de partició a partir de les àrees dels pics d’HPLC del compostos en octanol i en aigua després de la partició. Es va aconseguir un bon acord amb els valors de la literatura, mostrant la capacitat dels xips microfluídics per a una predicció precisa del coeficient de partició

    Characterisation of chitosan and its films for tissue engineering

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    Chitosan is a renewable polymer produced from a waste product of the seafood industry. It has been seen as useful for a range of applications due to its inherent properties. It is antifungal, antibacterial, biodegradable, biocompatible and has low immunogenicity which makes it attractive specifically for biomedical applications. Examples of chitosan’s possible applications include bioadhesive films, stem cell growth substrates and drug delivery agents. Chitosan is produced from the N-deacetylation of chitin. Chitin is the second most abundant polysaccharide in the world (by volume after cellulose) and is synthesized by many organisms which results in it being readily available, inexpensive and abundant. Its natural occurrence includes the shells of arthropods such as shrimps, crabs and the cell walls of yeasts. Unfortunately, due to its natural origin and the variation in processing conditions, chitosan is plagued by batch-to-batch variations which affect its widespread utilization. It thus requires appropriate characterization to allow exploitation of its inherent properties. The molecular structure of chitosan includes varying proportions of Dglucosamine and N-acetyl- D-glucosamine monomer units. The degree of acetylation (DA) is defined as the fraction of N-acetyl- D-glucosamine and the distribution of DAs is defined as its variation between polymer chains in a given sample. Although it has been well documented that a distribution of DAs exists (not all chitosan chains have the same DA), this is often overlooked. Therefore common characterization of chitosan is often incomplete and only takes into account one of the average DAs and neglects the distributions of DAs. The complexity and importance of the distribution of DAs had been revealed recently through a coupling of size-exclusion chromatography (SEC) with 1H NMR spectroscopy; however, it had not been measured before the work in this PhD. To allow an accurate characterization of polymers in solution, a true solution must be obtained. Unfortunately, the dissolution of chitosan is often overlooked. Utilizing capillary electrophoresis in the critical conditions (CECC), solution- and solid-state NMR spectroscopy, the dissolution of chitosan was probed. Obtaining a true chitosan solution has been proven to be challenging even with commonly used aqueous solvents. Aqueous AcOH which is most commonly used was seen to dissolve chitosan inefficiently compared to aqueous HCl. However, significant deacetylation was seen in chitosan dissolved in aqueous HCl and kept at high temperatures for prolonged periods of time. The standard for polymer size analysis, SEC, was shown to detect aggregation of the chitosan chains in the SEC eluent. Furthermore, comparisons of the average DA obtained with solution-state compared to solidstate NMR spectroscopy gave evidence of a clear bias in the characterization due to incomplete dissolution. This is extremely significant as chitosan is often characterized with solution-state NMR spectroscopy. The dissolution was concluded to be complex and a compromise is necessary in allowing a more complete dissolution and minimal deacetylation. However, for routinely measured average DA values, measurements should be undertaken in the solid state. To allow a more comprehensive characterization of chitosan composition, methods were developed in this PhD using free solution capillary electrophoresis in the critical conditions (CE-CC). CE-CC is a separation method and therefore is able to yield distributions. Complex polymers can have distributions of various parameters including composition, branching, end groups and molar mass. For chitosan, CE-CC separates by composition (or degree of acetylation). Capillary electrophoresis has been proven to be a robust technique for the separation and characterization of both natural and synthetic polymers. A method was developed to calculate dispersities from distributions obtained with CE-CC analogous to the calculation of dispersity from molar mass distributions determined by size-exclusion chromatography (SEC). Using a ratio of moments, the dispersity of electrophoretic mobility and composition distributions were obtained. The dispersity values represented either the heterogeneity of branching or composition of the complex polymers. This resulted in further characterization of complex polymers based on their composition or architecture. The dispersity values allowed the quantification and numerical representation of the heterogeneity. This allowed comparisons and trends to be quantified between samples. In the further analysis of chitosan, improvements in the separation were sought. This included changing the counter-ion of the buffer during the CE separation from sodium to lithium. Lithium showed trends of greater selectivity and combining these results with previous work in reducing the adsorption (lower pH and higher temperature) allowed a more accurate separation. The dispersity was then calculated for a larger range of chitosan samples and both distributions of electrophoretic mobilities and composition distributions were obtained. A trend was seen in which the dispersity first increased with the average DA and then began to reduce. Using the correlation between composition and mobility allowed composition distributions to be obtained for chitosan for the first time. This was the first determination of composition distributions and of their corresponding dispersity values for a statistical copolymer. The results identified chitosan samples with very similar measured average DA values having significantly different dispersity values. These results confirm the inaccuracy of characterizing chitosan by only through its average DA. Finally, to improve the use of chitosan for tissue engineering, regenerative medicine and other biomedical applications it was important to ensure low immunogenicity especially in the application of implantation. Poly(ethylene glycol), PEG, and the peptide RGDS was grafted onto the surface of chitosan and the chemical reaction was monitored using CE. The robustness of CE allows samples to be injected without sample preparation and allows it to be used effectively in the analysis of chemical reactions. The films were then characterized by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) and the grafting of PEG onto the surface of the chitosan film was validated. Previous cell attachment studies showed that the proliferation of cells occurred in specific regions. This was likely due to an inherent heterogeneity of the chitosan films which could be caused by incomplete dissolution and aggregation seen in the dissolution studies. To probe this heterogeneity of the chitosan films and powder, advanced solid-state NMR spectroscopy measurements were undertaken. The analysis compared the mobile and rigid fractions of chitosan. The results suggested similar behavior in both fractions, however, gave evidence of possible orientation of the acetyl group away from the backbone. The permeability of the films to small molecules was also tested and confirmed. In summary, the dissolution of chitosan was seen to be complex and currently used methods were either deemed inefficient in the dissolution or conversely caused degradation. A new method was developed to numerically represent the heterogeneity of composition or branching and this was tested with various complex polymer samples including chitosan. Further development of this method allowed composition distributions of a statistical copolymer (chitosan) to be obtained for the first time and the heterogeneity of composition to be obtained. New low immunogenicity films were produced by the grafting of poly(ethylene glycol) onto the surface of chitosan films and the grafting process was monitored by CE. The grafting was validated and the permeability and heterogeneity of chitosan films were also probed. Future work should involve probing the dissolution of chitosan with ionic liquids, applying the calculation of dispersity of both distributions of electrophoretic mobility and composition distributions to a broader range of polyelectrolytes, further improving the selectivity of the separation of chitosan and testing the biocompatibility of PEG grafted chitosan films. The methods developed in this thesis will enable chitosan to reach its potential for various applications ranging from tissue regeneration, through bioplastics to drug delivery

    Caracterización de una microemulsión de bromuro de tetradeciltrimetilamonio (TTAB) por cromatografía electrocinética de microemulsiones

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    Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2018, Tutors: Martí Rosés Pascual, Elisabet Fuguet JordàThe aim of this work is to characterize a tetradecytrimethylammonium bromide (surfactant), heptane (oil) and butan-1-ol (co-surfactant) microemulsion by microemulsion electrokinetic chromatography (MEEKC) through the solvation parameter model. This model is especially useful to describe the distribution of neutral solutes between two phases (the aqueous phase and the microemulsion). To do that, 69 neutral compounds with representative enough properties were analysed. The solvation parameter model is based on the linear free energy relationships (LFERs), which can be written as: log k = c + eE + sS + aA + bB + vV (3) where k is the MEEKC retention factor and E, S, A, B and V are the Abraham solute descriptors. The coefficients of the system (e, s, a, b and v) can be obtained by multiple linear regression and provide the properties of the studied microemulsion system. Once the coefficients of the system are determined, the studied system can be compared with other systems with known coefficients. In this work, the TTAB MEEKC system was correlated with octanol-water partition and SDS MEEKC systems. The coefficients are similar enough, thus the TTAB MEEKC system can be used to emulate the octanol-water partition. The octanol-water partition emulation is especially useful as it is a measure of the lipophilicity of compounds, which plays an important role in drug discovery and developmen

    Kidney cell electrophoresis

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    Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry

    Chiral separations using capillary electrophoresis

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