A series of microgels have been prepared by a surfactant free emulsion polymerisation based on an N-isopropylacrylamide (NIPAM) monomer. Poly(NIPAM) is a thermo sensitive polymer which undergoes a conformational transition close to the human skin temperature. Poly(NIPAM) was co-polymerized with hydrophobic butyl acrylate (BA) to provide a more favourable environment for drug molecules to partition into within the particle. A second co-monomer, acrylic acid (AA) was used to prepare pH/temperature sensitive microgels. The coupling reactions between microgels and cotton cellulose are only feasible if they both have appropriate functionalities. For microgels, this was achieved by using different initiators which introduce different functional groups on the particle surfaces and different surface changes. Cotton samples were successfully modified by carboxymethylation, periodate oxidation, grafting of 1,2,3,4-butanetetracarboxylic acid, and chloroacetylation in order to target possible reactions with the terminal functional groups of the microgel particles. Microgels were attached to the cotton fabrics using different methods. The maximum weight increase of cotton samples due to the attached microgels was 23.51 ± 0.29% (w/w). Drug loading and drug release were studied for free (unattached) microgels and novel wound dressings. Methyl paraben (MP) was used as a model drug compound. The results of MP release from free microgels show no significant influence of surface charge on MP release. However, BA-containing microgel particles release less MP than 100% poly(NIPAM) microgels due to hydrophobic interactions between the MP and the BA. Temperature-sensitive dressings release up to 45% more MP at 40°C than at 30°C. The drug release for pH-sensitive dressings was up to 24% higher at pH 5 than at pH 8. This demonstrates that the novel dressings can be classed as “smart” materials as they can respond to subtle changes in simulated wound fluid temperature and pH.

Intravenous lipid emulsions are used ubiquitously through the medical field as a source of parenteral nutrition. Development of new formulations requires an understanding of the metabolism of the product. Current methods of rate determination and of metabolism analysis have several drawbacks. Radioactive labelled assays have lower biological relevance, are time consuming due to separation steps and require long substrate preparation. Existing fluorescence assays based around triglyceride hydrolysis are impractical in emulsion systems due to high signal-noise ratio as well as the use of non-specific fluorescent dyes. Colorimetric methods such as the non-esterified fatty acids (NEFA) assay is expensive, requires multiple steps and specialised machinery. Due to the limitations of these techniques we developed a novel fluorescent assay using a lipoprotein lipase specific substrate incorporated into lipid emulsions. The lipoprotein lipase substrate, EnzChek® Lipase Substrate, green fluorescent, 505/515, is based on a triglyceride structure with a fluorescent dye at the Sn1 position and a dark quencher at the Sn2 position. LPL cleaves preferentially at the Sn1 position of triglycerides, which separates the dye from the quencher creating a fluorescent signal. The signal can then be detected using a fluorescent plate reader. Both lipid emulsion particles and EnzChek are substrates for LPL, so the hydrolysis of EnzChek is analogous for native emulsions particles. Over time, in the presence of LPL, fluorescent signal increases as more EnzChek is hydrolysed in tandem with emulsion particles. We have designed a range of emulsions with varied oil and surfactant composition. Using the EnzChek emulsion assay detailed above we are able to follow the rate of metabolism in real-time. This assay has been tested and found to be robust and reproducible. It is able to investigate differences in metabolism between Soybean oil, medium-chain triglyceride and fish oil emulsions. As well as changes in surfactant type and concentration.

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

Neste trabalho buscamos calcular as caracter´ısticas ´opticas e eletrˆonicas de nanofitas de di´oxido de estanho (SnO2) pura e com vacˆancia de oxigˆenio, e as modificac¸ ˜oes nas estruturas e propriedades, que ocorreram nas mesmas; utilizando como ferramenta os c´alculos de primeiros princ´ıpios, baseados no m´etodo da Teoria do Funcional da Densidade (DFT). Utilizamos o c´odigo SIESTA nas nossas simulac¸ ˜oes, utilizando algumas aproximac¸ ˜oes necess´arias, que ser˜ao descritas posteriormente. Atrav´es do SIESTA calculamos as func¸ ˜oes diel´etricas, partes real e imagin´aria, e por meio destas calculamos algumas propriedades ´opticas como coeficiente de absorc¸ ˜ao, refletˆancia, condutividade ´optica e outras. O estudo das propriedades eletrˆonicas das estruturas foi atrav´es das estruturas eletrˆonica de bandas, da densidade de estado total (DOS), da densidade de estado parcial (PDOS), do c´alculo do momento magn´etico resultante, e do estudo do mapa de contorno da diferenc¸a de densidade eletrˆonica. Os resultados obtidos se comparados a estudos experimental, mostram-se satisfat´orios, onde alguns resultados concordaram muito bem com outros trabalhos encontrados na literatura. / In this work we calculate the optical and electronic characteristics of tin dioxide nanoribbons (SnO2) and pure oxygen vacancy, and the changes in the structures and properties, which occurred in the same; using as a tool the first-principles calculations, based on the method Theory Density Functional (DFT). Using the SIESTA code in our simulations, using some approximations necessary, which will be described. Through the SIESTA calculate the dielectric functions, real and imaginary parts, and means calculate some optical properties such as absorption coefficient, reflectance and other optical conductivity. The study of the electronic properties of the structures was through the electronic band structure, the total state density (DOS) of the partial density of state (PDOS), calculating the resulting magnetic moment and of the study of contour map of the difference of electron density. The results are compared to experimental studies, appear satisfactory, where some results are in agreement with other studies in the literature. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

The modular polyketide synthase responsible for biosynthesis of the antibiotic mupirocin occupies 75 kb of Pseudomonas fluorescens NCIMB 10586, while a hybrid of PKS/NRPS is responsible for biosynthesis of the antibiotic thiomarinol located on a 97 kb plasmid pTML1 in Pseudoalteromonas spp SANK 73390. Biosynthesis of the acyl side chains in mupirocin and thiomarinol are thought to be either through esterification of the fully synthesised fatty acid (C\(_9\) or C\(_8\)) or through extension of the PK derived ester starter unit which is predicted to be carried out on MmpB and TmpB. mupU/O/V/C/F and macpE are proposed to be sufficient for the conversion of pseudomonic acid B to pseudomonic acid A. Mupirocin is regulated via quorum sensing, while regulation of thiomarinol was not identified. Production of thiomarinol was determined to occur after 8 hours of growth, while acidic conditions and use of acetone with ethyl acetate improved the extraction. TmlU, the thiomarinol amide ligase, did not complement a mupU mutant in mupirocin, and was found to block the biosynthesis of 9-hydroxynonanoic acid, causing truncation of 9-HN. This suggests that MupU, prevents MmpB from being an iterative PKS. KS-B2/ACP-B2 was shown to be involved in the removal of C8-OH from thiomarinol. Genetically manipulated mupU increased the production of mupirocin to 3 to 4 fold without abolishing PA-B production. Fused mupU-macpE complemented the NCIMB10586ΔmupUΔmacpE double mutant. However, insertion of this fusion into MmpB blocked the biosynthesis of mupirocin, while insertion after MmpA did not changed the pathway. Attempts to mobilise pTML1 revealed that a hybrid plasmid of RK2-R6K γ-ori was integrated into pTML1 but recovery of this cointegrate has not yet been recovered in E. coli.

Haemorrhage remains a leading cause of death in civilian and military environments. Recent research into emergency treatments for severe haemorrhaging injuries has resulted in production of a number of advanced haemostatic products. In certain scenarios, concomitant release of toxic materials may exacerbate trauma and ultimately reduce survival from such injuries. In particular, systemic absorption of the nerve agent VX (S-[2-(diisopropylamino)ethyl]-O-ethyl methylphosphonothioate) via wounds may rapidly cause muscle paralysis and death. This research explored the hypothesis that a haemostatic product could perform an additional function as a wound decontaminant. […] In summary, this project has successfully demonstrated that haemostatic products, based on an adsorptive mechanism of action, offer a highly effective means of countering the rapidly fatal effects of the chemical warfare agent VX present in wounded skin tissue. Further development of the concept of a haemostatic decontaminant has life-saving implications.

An analysis of over 34,000 free-text messages assigned by pharmacists to prescription orders over a 12-month period showed a sub-optimal exchange of information with the physician. Focus groups and observational research were conducted to provide a more in-depth understanding of the factors involved. The use of CPOE did not reduce opportunities for personal interaction. The capability to communicate electronically facilitated a non-interruptive workflow, beneficial for staff time and for limiting distractions. It also improved clinical documentation, which helped coordinate care of patients between members of the pharmacy team. However, the research identified several barriers to the effectiveness of communication via the CPOE system, including: the increased frequency of messages sent; poor display characteristics of the message; poor access to information to inform decision-making; one-way communication; and no assigned responsibility to respond. These factors need to be considered in the design of systems and supported by interprofessional training to optimise communication between the professionals.

There is a clear and well-established need for new antibiotics. The biosynthesis of the antibiotic mupirocin is an important model trans-AT polyketide synthase pathway. A key tailoring step is the removal of a hydroxyl from the intermediate pseudomonic acid B (PA-B), which generates active pseudomonic acid A (PA-A). This is proposed to occur on discrete proteins after release from enzyme MmpB. In this work, a systematic screen for genes required for this key step was developed, which implicated ten genes. On a multi-gene expression plasmid, these ten genes encoded all functions necessary for conversion. This re-identified seven genes from the existing proposed model. A novel requirement for MupM, MupN, and the DNA sequence but not protein function ofMupL was uncovered. This represents the first identification of all genes required for this surprisingly convoluted pathway. With the aim of re-engineering the location of these tailoring steps, domains from thiomarinol enzyme TmpB were inserted into MmpB. Low-level conversion to PA-A was achieved using thiomarinol tailoring enzymes. The TmpB KS0 domain was used to load the normally free-standing MacpE, and high-level conversion was achieved on the hybrid enzyme. This success opens the future path to further MmpB modifications to generate mupirocin derivatives.

Polyketide synthases (PKS) are enzyme complexes that synthesise many natural products of medicinal interest, notably a large number of antibiotics. The present work investigated the mupirocin biosynthesis system, comparing it with similar pathways such as thiomarinol and kalimantacin. The focus was on the structural modelling of the protein complexes involved in antibiotic synthesis, via molecular simulation and the analysis of structural and sequence data. Structural docking of acyl carrier proteins (ACP) cognate for an HMG-CoA synthase orthologue responsible for β-methylation (MupH) identified key residues involved in the recognitions specificity of the interacting partners, further supported by mutagenesis experiments, which thus allows prediction of β-methylation sites in PKS. Moreover, complementation and mutagenesis experiments performed on MupH homologs from kalimantacin and thiomarinol systems suggests specificity between the ACP:HCS proteins in the β-branching suggesting the possibility of engineering multiple specific β-branching modifications into the same pathway. Molecular dynamics simulations of ACPs from the mupirocin cluster revealed that the PKS ACPs form a cavity upon the attachment of the phosphopantetheine and acyl chains similar to what is seen in the fatty acid synthase ACPs and provide a better understanding of the structure function relationship in these small proteins. Molecular docking of the putative cognate substrate with the ketosynthase (KS) homo dimer of module 5 of the MmpA in the mupirocin pathway revealed a loop that may control specificity for the α-hydroxylated substrate and mutagenesis experiments support this proposition.