1,720,959 research outputs found
Aggregation Prone Region analogues: synthetic excipients for protein formulation
Get PDFIn the last few decades, protein therapeutics have become a relevant segment of the pharmaceutical industry. However, clinical use of biotherapeutics can be limited by poor stability of proteins in the different steps of manufacturing, storage and formulation, with subsequent formation of protein aggregates. Aggregation affects not only therapeutics proteins, with detrimental effects on production costs, final product yields and therapeutic efficacy, but also endogenous proteins, as it leads to the formation of large aggregates deposits which have been correlated to different neurodegenerative diseases. Consequently, a range of stabilisers have been developed to increase the stability of biotherapeutics in formulations, or prevent the aggregation of endogenous proteins. Among the different strategies employed, excipients that stabilise proteins through non-covalent interactions have been reported.
This thesis focusses on the stabilisation of proteins through hydrophobic interactions: here, the synthesis of short, hydrophobic, stabilisers is reported. These stabilisers were tested on different proteins, to interact with hydrophobic patches on proteins primary strucures, and block these patches from mutual, self-interactions that may lead to proteins aggregation.
In the first part of this work, peptides analogous of hen egg lysozyme Aggregation Prone Region (APR), were synthesised, and tested on lysozyme to verify any potential interaction between these synthetic peptides and their homologous sequence on lysozyme, to block its site from self-interactions that lead to aggregation. For improved solubility and to enhance its stabilising effect, the APR peptide fragment was finally copolymerised with monomer N-hydroxyethylacrylamide, to generate a peptide-polyacrylamide copolymer stabiliser. Pleasingly, the copolymer proved to be able to delay the onset of lysozyme aggregation, which was induced in strong basic conditions.
Encouraged by these results, in the second experimental chapter this strategy was expanded by developing a library of amphiphilic block copolymers, comprising hydrophobic amino acid-like moieties, potentially able to non-covalently interact with hydrophobic, self-aggregating protein domains, and prevent protein aggregation/denaturation. Three moieties were chosen, indole 3-acetic acid, phenyl acetic acid and methylisobutiric acid, to mimic the side chains of three amino acids, tryptophan, phenylalanine and isoleucine, respectively. The copolymers were tested on two different proteins, hen egg lysozyme, bovine pancreatic insulin, and the antimicrobial peptide IDR 1018. Potential interaction between the proteins and the copolymers was evaluated under stressful conditions, which induced proteins aggregation, measured by turbidity and solubility studies. Promising stabilising effects were shown by some of the indole-contaning copolymers, which proved to be able to prevent the aggregation and to increase the solubility of both insulin and peptide IDR 1018. Hydrophobic Indole-based oligomers were further tested to evaluate their efficacy in encapsulating the antimicrobial peptide IDR 1018. The peptide was first ion paired with the antimicrobial molecule usnic acid, to develop a hydrophobic complex for enhanced IDR 1018 encapsulation and potential co-delivery of two antimicrobial drugs.
In the fourth experimental chapter of this thesis, cholanic-polyacrylamides conjugates were synthesised for potential non-covalent protein conjugation. Cholanic acid has been previously investigated for its ability to interact with proteins hydrophobic patches. In particular, a series of PEG-cholanes of different molecular weight were used to efficiently complex two different proteins, the recombinant human growth hormone (rh-GH) and the recombinant human granulocyte colony stimulating factor (rh‐G‐CSF). improving their bioavailability and extending their half-life. Here, cholanic acid was incorporated into a RAFT agent and used to mediate the polymerization of N-hydroxyethylacrylamide, to develop cholanic-polyacrylamides of different length. The polymers were successfully employed as protein complexing agents for two model proteins, bovine serum albumin and bovine pancreatic insulin.
Finally, the last chapter is presented in a form of a draft paper, and is part of a collaborative work started by a former PhD student in our group, Joao Madeira do O. A series of linear and 4-arm glycopolymers, were previously prepared by copper azide alkyne cycloaddition (CuAAC) functionalisation of preformed poly(propargyl methacrylate)s with different sugar azides. In this thesis, the reversible, non-covalent interaction between the small hydrophobic molecule Nile Red and linear and 4-arm glycopolymers was evaluated. Results suggest that the interaction occurs between the dye molecule and single polymer chains, suggesting that these glycopolymers do not self-assemble in supramolecular aggregates and act instead as unimolecular micelles
Aggregation Prone Region analogues: synthetic excipients for protein formulation
Get PDFIn the last few decades, protein therapeutics have become a relevant segment of the pharmaceutical industry. However, clinical use of biotherapeutics can be limited by poor stability of proteins in the different steps of manufacturing, storage and formulation, with subsequent formation of protein aggregates. Aggregation affects not only therapeutics proteins, with detrimental effects on production costs, final product yields and therapeutic efficacy, but also endogenous proteins, as it leads to the formation of large aggregates deposits which have been correlated to different neurodegenerative diseases. Consequently, a range of stabilisers have been developed to increase the stability of biotherapeutics in formulations, or prevent the aggregation of endogenous proteins. Among the different strategies employed, excipients that stabilise proteins through non-covalent interactions have been reported.
This thesis focusses on the stabilisation of proteins through hydrophobic interactions: here, the synthesis of short, hydrophobic, stabilisers is reported. These stabilisers were tested on different proteins, to interact with hydrophobic patches on proteins primary strucures, and block these patches from mutual, self-interactions that may lead to proteins aggregation.
In the first part of this work, peptides analogous of hen egg lysozyme Aggregation Prone Region (APR), were synthesised, and tested on lysozyme to verify any potential interaction between these synthetic peptides and their homologous sequence on lysozyme, to block its site from self-interactions that lead to aggregation. For improved solubility and to enhance its stabilising effect, the APR peptide fragment was finally copolymerised with monomer N-hydroxyethylacrylamide, to generate a peptide-polyacrylamide copolymer stabiliser. Pleasingly, the copolymer proved to be able to delay the onset of lysozyme aggregation, which was induced in strong basic conditions.
Encouraged by these results, in the second experimental chapter this strategy was expanded by developing a library of amphiphilic block copolymers, comprising hydrophobic amino acid-like moieties, potentially able to non-covalently interact with hydrophobic, self-aggregating protein domains, and prevent protein aggregation/denaturation. Three moieties were chosen, indole 3-acetic acid, phenyl acetic acid and methylisobutiric acid, to mimic the side chains of three amino acids, tryptophan, phenylalanine and isoleucine, respectively. The copolymers were tested on two different proteins, hen egg lysozyme, bovine pancreatic insulin, and the antimicrobial peptide IDR 1018. Potential interaction between the proteins and the copolymers was evaluated under stressful conditions, which induced proteins aggregation, measured by turbidity and solubility studies. Promising stabilising effects were shown by some of the indole-contaning copolymers, which proved to be able to prevent the aggregation and to increase the solubility of both insulin and peptide IDR 1018. Hydrophobic Indole-based oligomers were further tested to evaluate their efficacy in encapsulating the antimicrobial peptide IDR 1018. The peptide was first ion paired with the antimicrobial molecule usnic acid, to develop a hydrophobic complex for enhanced IDR 1018 encapsulation and potential co-delivery of two antimicrobial drugs.
In the fourth experimental chapter of this thesis, cholanic-polyacrylamides conjugates were synthesised for potential non-covalent protein conjugation. Cholanic acid has been previously investigated for its ability to interact with proteins hydrophobic patches. In particular, a series of PEG-cholanes of different molecular weight were used to efficiently complex two different proteins, the recombinant human growth hormone (rh-GH) and the recombinant human granulocyte colony stimulating factor (rh‐G‐CSF). improving their bioavailability and extending their half-life. Here, cholanic acid was incorporated into a RAFT agent and used to mediate the polymerization of N-hydroxyethylacrylamide, to develop cholanic-polyacrylamides of different length. The polymers were successfully employed as protein complexing agents for two model proteins, bovine serum albumin and bovine pancreatic insulin.
Finally, the last chapter is presented in a form of a draft paper, and is part of a collaborative work started by a former PhD student in our group, Joao Madeira do O. A series of linear and 4-arm glycopolymers, were previously prepared by copper azide alkyne cycloaddition (CuAAC) functionalisation of preformed poly(propargyl methacrylate)s with different sugar azides. In this thesis, the reversible, non-covalent interaction between the small hydrophobic molecule Nile Red and linear and 4-arm glycopolymers was evaluated. Results suggest that the interaction occurs between the dye molecule and single polymer chains, suggesting that these glycopolymers do not self-assemble in supramolecular aggregates and act instead as unimolecular micelles
Direct routes to functional RAFT agents from substituted N-alkyl maleimides
Get PDFN-substituted maleimides have become an indispensable tool for the synthesis of bioconjugates and functional materials. Herein, we present three strategies for the incorporation of N-alkyl substituted maleimides into RAFT agents and show that these maleimide-derived CTAs can be used to easily introduce a range of chemical functionality at the β-position of polymer chains, resulting in α,β,ω-functional RAFT polymers. With both functional maleimides and RAFT agents that are increasingly available on the market, the approach presented in this study could facilitate the synthesis of end-functional macromolecules and will complement well the range of existing synthetic routes, including those utilising N-substituted maleimides, to functional polymeric materials
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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