Proteins are large flexible molecules and conformational dynamics is one of their fundamental properties which correlate the protein’s structure and function [1] [2]. The crystal structure of biomolecular systems such as enzymes reveals the important atomistic details in terms of the ligand binding and possible mechanism albeit providing no information about how conformational flexibility and dynamics influences the protein structure and its key determinants. There is no information regarding the electronic structure and the chemically relevant components of the enzymes and how the protein environment affects the electronic structure. In order to provide understanding of how the conformational flexibility influences structure-function relationships of the enzymes, we applied classical molecular dynamics simulations using Gromacs [3] [4] [5] and Amber [6] [7] packages. The effect of the protein environment on the electronic structure of the active site were studied using Quantum Mechanics and Molecular Mechanics (QM/MM) [8] using ONIOM [9] [10, 11] implemented in Gaussion09 [12] [13]. Tyrosylproteinsulfotransferase (TPST): TPSTs catalyze the transfer of negatively charged sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue of polypeptide to form a tyrosine O4-sulfate ester [14]. The binding of the substrate peptide showed more open conformation in Tyrosylproteinsulfotransferase-2 (TPST-2) enzyme in contrast to the crystal structure [15] [16]. There were identification of new hydrophobic interactions responsible for the stabilization of the enzyme dimer [16]. The binding of the substrate and cofactor to the apoenzyme contributed to the stability of the whole active complex, influenced the local interactions in the binding site and importantly, affects the pattern of the correlated motions in the entire molecule [16]. NirE an S-adenosyl-L-methionine dependent Methyltransferase: The NirE enzyme catalyses the transfer of a methyl group from the S-adenosyl-L-methionine (SAM) to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. The binding of the substrate contributes to the stabilization of the structure of the full enzyme complex [17]. The conformational changes influence the orientation of the pyrrole rings of the substrate [17]. The mutations of binding and active site residues leads to sensitive structural changes which influence binding and catalysis [17]. Matrix metalloproteinase-1 (MMP-1): The molecular dynamics studies on the Matrix metalloproteinase-1 (MMP-1) were in good agreement with the experimental observation that in the MMP-1•THP (Triple Helical Peptide) X-ray crystallographic structure MMP-1[18] is in a "closed" conformation [19]. The interactions of the THP with both the CAT and HPX domains of MMP-1 are dynamic in nature, and the linker region of MMP-1 influences the interactions and dynamics of both the CAT and HPX domains and collagen binding to MMP-1 [19]. The mutations in the MMP-1 have distinct impact on the correlated motions in the MMP-1•THP. An increased collagenase activity corresponded to the appearance of a unique anti-correlated motion and decreased correlated motions, while decreased collagenase activity corresponded both to increased and decreased anti-correlated motions. Non-heme Fe2+ and 2-oxoglutrate (2OG): The non-heme Fe2+ and 2-oxoglutrate (2OG) dependent dioxygenases such as FTO, AlkB, PHF8 and KIA1718 perform important tasks in homeostasis through the methylation of DNA and histone proteins. The linker region shows increase conformational flexibility and dynamics in PHF8 and KIA1718 and is important for the catalysis. The jelly roll motif structure also showed conformational stability for all demethylases and indicates its vital role in maintaining the iron geometry in the active site. The N domain of the FTO enzyme and the L1 loop region showed increased conformational flexibility and dynamics. The QM/MM optimized structure of reactant complex showed the effect of the conformational flexibility. An important insight into the structure function relationship of different enzymes has been obtained by applying a large number of Atomistic Molecular Dynamics Simulations and Quantum Mechanics/Molecular Mechanics to different enzymes which cannot be gained experimentally. The effect of conformational dynamics, flexibility and important interactions of the active site residues can be used in chemical biology and biotechnology for structure based drug design and in the engineering of novel biocatalyst.

Although it is known that DNA topo IIβ is required for the regulation of transcription during neural development and differentiation, it is not clear whether the enzyme is required during differentiation of human monocytes into macrophages and/or the subsequent transcription of cytokine genes. To test this, a robust model of differentiation of monocyte-like cells into macrophage-like cells using U937 and HL-60 cells treated with phorbol 12-myristate 13-acetate (PMA) and Lipopolysaccharide (LPS) was validated. Differentiation was determined by morphological and growth characteristics and CD11b surface antigen expression as determined by flow cytometry. qRT-PCR was also used to measure mRNA transcript levels of key genes known to be up-regulated during monocyte differentiation and the secretion of pro-inflammatory cytokines produced by differentiated cells were measured using ELISA. siRNA topo IIβknockdown did not hinder monocyte-like cells from undergoing differentiation, however experiments revealed a correlation between topo IIβknockdown and secreted TNFα, with the latter decreasing when topo IIβwas reduced. This pattern was also noted when measuring IL-1βsecretion. Similar results were seen using a Murine transgenic fibroblast cell line lacking topo IIβ, which when stimulated with LPS secreted significantly lower levels of IL-6 compared to the wild type cells. Thus topo IIβexpression is necessary for secretion of normal levels of the cytokines, TNFα, IL-1βand IL-6 in response to LPS at certain time points. In addition in the macrophage-like state of the two cell lines, the relative levels of the βisoform (mRNA and protein) were shown to be significantly increased compared to α, further outlining the importance of topo IIβin the differentiated state. Chromatin immuno-precipitation followed by qPCR showed however that topo IIβwas not associated at three defined proximal promoter regions of either the TNFαand IL-1βgenes, although further studies are required to rule out a direct association of topo IIβwith these and other regions of the genes. Down regulation of topo IIβprotein using the inhibitor ICRF-193 did not hinder monocyte-like cells from undergoing differentiation either. However, contrary to the knockdown results, a 6 h pre-treatment with 1 nM ICRF-193 increased TNFαlevels in these cells, both at the mRNA and the protein level, along with a slight increase in secreted TNFα. NF-κB, EGR2, TLR4 and TLR2 transcript levels were also increased under these conditions. Thus further studies are required to determine if these increases are due to additional cellular effects of the drug or whether topo IIβmay play an inhibitory effect on transcription. Thus it is clear that topo IIβplays an important role in expression of cytokines and understanding the exact nature of this requires further research that may yield potential new avenues for treatment of disease.

Foot pain and pathology can be disabling leading to more complex orthopaedic complaints over time. Footwear is often attributed as a significant factor in the development and persistence of foot pain, yet little is known about the impact everyday footwear choice has on the development of these pathologies and foot biomechanics. The aim of this collection of work is to assess the impact footwear choice has on foot biomechanics. A mixed methods approach has been employed across various publications to investigate the following; choices made when purchasing footwear, the impact footwear structure and styling has on foot mechanics and comfort and the effect of unstable shoes on muscle function. The publications employed literature reviews, qualitative questionnaires, repeated measures and quasi-experimental designs to address the research questions. There is a paucity of research regarding the effects that everyday footwear have on the feet of healthy individuals. A flat ballet pump was found to be the primary shoe of choice for young females with the colour of trainers being selected by sport science students. Altered physical characteristics of the shoe caused elevated dorsal and plantar foot pressure, impaired comfort and altered function. Fashionable exercise shoes were shown to demonstrate a varied effect on muscle activity. The availability of suitably fashionable and functional footwear appears to be severely limited leading to consumers purchasing inappropriate and ill-fitting footwear that may contribute to foot pathology. An extensive review of design, properties and manufacture with specific consideration to pathology in the footwear industry is recommended to improve footwear choice. The publications presented add new knowledge when evaluating consumer choice of footwear and the potentially adverse impact popular female fashion shoes have on foot biomechanics. The results also contribute to a wider understanding of the impact everyday footwear has on foot pathology and help in the application of footwear related treatment and rehabilitation plans.

No description available.

The main objective of this research programme was to determine the factors influencing the decolourisation of dyes at low pH by different peroxide species, both in the presence and absence of metal ion catalysts and, therefore, to find a set of optimal conditions for application to wastewater treatment processes. An additional study looked at whether peroxoborates were capable of acting as nucleophiles. The specific aims of the study were: to investigate the in-situ formation of peracetic acid from the equilibrium formed between hydrogen peroxide and acetic acid, and whether this can be achieved without the addition of an acid catalyst such as sulphuric acid; to study the comparative reactivity of in-situ generated peracetic acid and hydrogen peroxide towards a range of dyes used in industry; to investigate the catalytic potential of a range of metal ions towards the reaction between peroxides and dyes; to investigate the structural features of dyes that might influence reactivity (decolourisation); and to investigate the reactivities of other peracid-like peroxide species that can be generated from hydrogen peroxide (peroxoborates and peroxocarbonates).The novel aspects arising from this study were: (a) The development of a new method for the in-situ generation of peracetic acid that gives the same equilibrium yield as established methods yet does not require the addition of an acid catalyst;(the reaction was slow, but there was minimal decomposition and so it is ideal for circumstances that allow the preparation of peracetic acid well in advance of use). (b) The first comprehensive study of the bleaching potential of peracetic acid and hydrogen peroxide towards a wide structural range of dyes both in the presence and absence of metal ions (iron, manganese, silver and copper). (c) The inference that for iron-catalysed bleaching of azo dyes by peracetic acid the catalytic mechanism involves pre-complexation of iron and dye, followed by reaction of the 'activated' complex with peracetic acid rather than a free radical mechanism that might have been expected for such systems. (d) The evidence that, in contradiction to literature studies, peroxoborate species do not act as nucleophiles. As an introduction to this work the reactions of peroxyacids are described in general terms. The experimental work is divided in three parts. In Chapter two, the homogeneous preparation of peracetic acid (PAA) from acetic acid (AA) and hydrogen peroxide (H2O2) was investigated with and without the catalysis of sulphuric acid (H2SO4). The formation of PAA and total peroxide content was determined by iodimetric titration. The reaction was slow in the absence of a strong acid catalyst, and was faster with a sulphuric acid catalyst. There was no loss of total peroxide over the timescales of both reactions, whether a catalyst was used or not. The equilibrium constant for peracetic acid formation at temperature of 20 was found to be 2.04 with a catalyst, and 2.10 without catalyst. The rate constant for the hydrolysis of peracetic acid for both forward and reverse reactions increased when the sulphuric acid concentration was increased from 0.02 M to 0.32 M. Linear relationships were found between the observed rate constants and H+ concentrations at 25oC. Moreover, it was found that the preparation of peracetic acid showed a first-order dependence with respect to peroxide concentration. In Chapter three, the application of this preparation of peroxyacids to the degradation of different types of dyesstuffs was investigated. As we know, physical or other chemical methods for dye degradation are expensive and can generate secondary pollution. In this part of the study the reactions of dyes with hydrogen peroxide and peracetic acid in the absence and presence different metal ions (Fe3+, Cu2+, Mn2+ and Ag+) were investigated. The iron/peroxyacid system was found to be the most effective. Consequently, Chapter 4 evaluates the decolourization of five azo dyes under conditions of bleaching by peracetic acid in the presence of Fe3+ as a catalyst. The experiment was carried out in aqueous acidic media. Dye oxidation systems are complex because: they involve several different tautomers; there is the possibility of dye aggregation at lower dye concentrations; and the oxidant species involved can be either the undissociated peroxide acting as an electrophile, or the dissociated peroxide acting as a nucleophile. The results obtained for the reaction of azo dyes with peracetic acid without added iron, when converted to the second order rate constant for the electrophilic reaction, k2E gave a value of 4.5x10-6 dm3 mol-1 s-1 for orange II, which is very high. This may be due to trace metal ions still being present and catalysing the reaction, possibly from impurities in the dye itself. No metal ion chelators were used in the present study because the bulk of the study was designed to elucidate the effect of metal ions concentration on reaction rate. For the catalysed reactions a significantly increased rate of absorbance decrease with increasing iron concentration was observed. Saturation of iron was also demonstrated at high iron concentrations, suggesting the formation of an iron (III)-dye complex which then reacted with peracetic acid. The maximum rate of reaction was observed at an iron concentration of 0.012 M, and the results showed a reactivity order of Ponceau 4R > Amaranth > (Orange II & Carmosine) > Black PN; Orange 1 was unreactive under these conditions. Also one of the key objectives of this chapter was to determine the optimum conditions for dye degradation in terms of pH and oxidant and catalyst concentrations. The optimum conditions for maximum degradation occurred at the highest pH of 3.0 and at about 1x10-3 M iron. Evidence of the possible involvement of radicals in our studies comes from the observation of a lag phase followed by a more rapid bleaching phase in the oxidation of azo dyes by peracetic acid at the lowest iron concentrations (another possibility is that at these iron concentrations the reactive iron complex forms at a much slower rate). However, this process is slow by comparison with the rate of oxidation at higher iron concentrations that do not exhibit this lag phase; consequently, if free radical mechanisms are suggested then they are not significant compared to the proposed formation of a reactive iron-dye complex.ixThe work contained in final experimental Chapter aimed to clarify whether or not any of the peroxyborate species displayed nucleophilic characteristics and thus accelerated the rate of the reaction of hydrogen peroxide with p-nitrophenyl acetate. The pH range of 6.0 to 8.0 is critical in terms of the distribution of peroxide species for a hydrogen peroxide / boric acid system. The triganol peroxoboric acid, B(OH)2OOH, is the only significant peroxoborate species below pH 6.5. However, above this pH, increased concentrations of the monoperoxoborate anion, B(OH)3OOH, the peroxodiborate anion, (HO)3BOOB(OH)32-, and the diperoxodiborate anion (HO)2B(OO)2B(OH)22-, are formed, with diperoxoborate, B(OH)2(OOH)2- forming at higher hydrogen peroxide concentrations. Therefore this is the ideal pH range in which to elucidate any effects of borate on the reaction of hydrogen peroxide and PNPA. The observed second order rate constants (k2obs) for the reaction between p-nitrophenyl acetate and hydrogen peroxide, and the corresponding second order rate constants, k2, for the reaction of the perhydroxyl anion with p-nitrophenyl acetate was determined by equation:In borate buffer the k2 values were significantly reduced compared to other buffers; this reduction was consistent with the hydrogen peroxide complexing with borate to form a range of non-reactive (towards carbonyl groups) peroxoborate species, thus also reducing the equilibrium concentration of the perhydroxyl anion. There was no evidence for peroxoborate species that could act as nucleophiles, in contradiction of literature claims. Values of k2 in the case of phosphate buffer compared reasonably well with values in the literature of 3140 and 3520 dm3 mol-1 s-1 obtained at pH 6.8 in ionic strengths of 0.02 dm3 mol-1 and 0.1dm3 mol-1 respectively. In carbonate buffer the literature value is 3785 dm3 mol-1 s-1 at pH 10, ionic strength 0.1 M, in borate buffer.

The Maptaphut Industrial Estate is located on the Gulf of Thailand, Rayong Province. The area, which has been designated as a main centre for the petrochemical industry currently occupies 16 sq km and comprises petrochemical plants, chemical and fertilizer plants, refineries, construction plants, and steel industry; there are also residential and commercial areas (IEAT, 2004). There is a significant population around the site, with 24,000 inhabitants in the immediate vicinity according to Jadsri et a/ (2006). The estate has been held responsible for deaths and hospital admissions due to leaks and accidents dating back as far as 1997. Whilst the environmental and health and safety performance of the estate as a whole has significantly improved over recent years, there are still significant outpatient admission rates to Maptaphut hospital for respiratory illness, as recently reported by Jadsri et al. (2006), raising the question of whether local emissions are significantly contributing to ill health, or whether general background concentrations of pollutants from nearby road sources and from Rayong City are the main contributions. The main aim of this research, therefore, was to accurately model the dispersion of pollutants from the estate, and to attempt to quantify the health impacts of these emissions. The specific objectives of this study were to (a) to characterise meteorological conditions in the Maptaphut area; (b) to develop a multiple linear regression statistical model to characterise and predict atmospheric pollutant concentrations in Maptaphut; (c) to investigate the relationship between air pollution and ill health in Maptaphut using a multiple linear regression statistical model; (d) to evaluate the effectiveness of Gaussian and Computational Fluid Dynamics atmospheric dispersion modelling software packages in predicting ground level pollutant concentrations at points around the industrial estate and (e) to use the results of the dispersion modelling studies to assess the contribution of the industrial estate to the overall atmospheric pollutant load in the Maptaphut area, and from published health impact factors, to assess the overall health impact of the estate. The first objective was to characterise the environmental status, trend, and impacts of air pollution during the period 1998 to 2007. The estate is located in the coastal area; thus, the role of the sea-land breeze has a significant role in the dispersion of air pollutants harmfulness. Data collected for the Maptaphut Industrial Estates area, including regional, temporal and spatial considerations included: meteorological data from 100-metres tall meteorological mast; ambient air quality data from three ambient air quality monitoring stations; industrial emissions data; traffic volume on nearby major roads; and outpatient admissions data at the Maptaphut and Rayong hospitals. Comparisons with the ambient air quality in the Bangkok area were made, and the daily and yearly trends in concentrations of the main air pollutants were analysed. Multiple linear regression models correlating pollutant concentrations with respiratory outpatient admissions rates showed that 03, PMio and NO were statistically significant determinants. The overall correlation had a coefficient of Determination (R2) of 41.4% for one week average data, increasing to 51.2% when air temperature and %RH were included. Accumulation effect of pollutants up to four weeks period exposure does not appear to have an effect. A basic health impact analysis study using the ADMS modelled concentrations and the WHO AirQ tool, along with default risk factors, showed that emissions from the Maptaphut industrial estate account for almost all of the NO2 and SO2 related respiratory illness and between 10 and 27% of the PMio related admissions; this actually represents less than 2% of the total respiratory admissions for this area.

Bacteroides fragilis NCTC 9343 is a Gram-negative anaerobic bacterium with genomic DNA of 5205 Kb and a GC ratio of 43%. It is a commensal organism that can act as an opportunistic pathogen and is commonly present on the mucous membranes. It causes a variety of infections including intra abdominal infections, perirectal abscesses and decubitus ulcers. Enterotoxigenic forms are capable of causing diarrhoea in children and animals. Enterobacter cancerogenus ATCC 35316 is also a Gram-negative facultatively anaerobic bacterium with genomic DNA of 4602 Kb and a GC ratio of 55%. It is a naturally occurring human gut symbiont known to exhibit resistance to antibiotics like aminopenicillins. It has also been reported in cases of severe osteomyelitis and infections of bones and joints. This study aims to analyse the differential expression of proteins in the presence of mucin since it serves as the first site of adherence for the bacteria. The E. cancerogenus and B. fra gilis proteins were extracted and separated by two dimensional electrophoresis from logarithmic phase cultures grown in semi-defined media enriched with or without porcine gastric mucin Types II and III. The gel images were analysed using Bio-Rad PDQuest, Ludesi Redfin and Nonlinear Dynamics SameSpots softwares. It was observed that the presence of mucin in the media affected the expression of a number of proteins in E. cancerogenus and B. fragilis cells. The protein spots of interest were excised, hydrolysed using trypsin and subjected to electrospray ionisation based LC-MS analysis in order to determine the identity of the digested proteins and obtain a better understanding of the interactions of B. fra gilis and E. cancero genus with mucin. The outer membrane protein surface antigen X was found to be up-regulated in both mucin Type II and III enriched media in E. cancerogenus. Some of the other proteins that were differentially regulated in both E. cancerogenus and B. fra gilis included the elongation factor Ts, malate dehydrogenase, triose phosphate isomerase and thiol peroxidase proteins indicating that these proteins may be associated with the ability of bacteria to grow in mucin and may be potential virulence factors. Genes encoding the proteins CAH06598 and CAH09443 from the glycoside hydrolase families 95 and 97 in B. fra gilis strain NCTC9343 were cloned, overexpressed and purified using nickel affinity and gel filtration chromatography. The enzymes were found to be active by performing fluorimetric assays using methyl-umbelliferyl sugar substrates. Diffracting crystals of CAH09443 were obtained from the PACT ANION screens containing polyethylene glycol and sodium malonate as a precipitant. Structure determination was achieved via molecular replacement using the glycoside hydrolase Family 97 α-galactosidase, BtGH97b, from Bacteroides thetaiotaomicron as a starting model. The structure of CAH09443 was shown to be composed of a N-terminal β-super-sandwich domain and a canonical (β/α)₈ barrel, similar to the two other glycoside hydrolase family 97 enzyme structures reported.

The last fifty years has seen an increase in the production of synthetic or artificial enzyme substrates used to identify and quantify enzymes. These substrates have found applications in a range of biomedical science disciplines. Used in biochemistry and clinical chemistry to identify and measure enzymes, some of these substrates have been adapted for use in microbiology, particularly bacterial diagnosis and, in more recent years, molecular biology. The use of artificial chromogenic and fluorogenic enzyme substrates to identify certain bacteria is now common place in medical laboratories worldwide. Not all bacteria can be identified with existing and commercially available artificial substrates. Some of these can be slow to yield results, imprecise, expensive or require a technical method too complicated to provide a viable laboratory test. Therefore, the search for new, more efficient, biochemical tests has progressed, with novel substrates and inventive applications being developed continually. In this study, core compounds were synthesised by various condensation reactions and their characteristics evaluated with respect to colouration/fluorescence and possible enhancement of these properties by metal chelation. Promising candidates were selected for glycosidation, via modified Koenigs-Knorr reactions, in an attempt to synthesise artificial substrates. Several commercially available core molecules were also subjected to glycosidation. The more successful substrates included glycosides of alizarin, nitrosalicylaldehyde and 3- hydroxyflavone. The galactoside of nitrosalicylaldehyde was evaluated in solid agar media and found to be selective for certain Gram-negative bacteria. When similarly investigated, the 3- hydroxyflavone-β-D-glucoside showed the possibility of being used in a procedure for the isolation of the clinically significant pathogens including Listeria monocytogenes. The enzyme kinetics of β-glucosidase with this substrate were also determined in a novel fluorescence assay and compared favourably to the well documented 4-methylumbelliferyl-β-D-glucopyranoside. Alizarin-2-yl-β-D-galactoside and p-naphtholbenzein-β-D-galactoside were successfully utilized for the screening of recombinant and non-recombinant Escherichia coli transformants produced routinely in molecular biology. Aminopeptidase substrates have been shown to be useful for the detection of enzymes which hydrolyse peptides that are specific to certain bacteria. To allow the evaluation of novel aminopeptidase substrates, that were to be subsequently synthesised, a cost effective, large scale source of recombinant leucyl aminopeptidase enzyme was developed via gene cloning techniques. Consequently, the products of this study may serve a beneficial purpose in future enzymatic investigations, medical diagnosis and molecular biology.

The development of novel molecular tools has provided the scientific community with quick, easy, and scientifically sound ways of identifying individual strains belonging to the Burkholderia cepacia complex (Bcc). Bcc strains isolated from the sputum of 44 patients attending the Freeman Hospital Cardiopulmonary Transplant Unit were genotyped using recA PCR-RFLP analysis, and a clonality study performed using PFGE analysis. It was found that B. cenocepacia and B. multivorans were the predominant colonizing strains in these patients, and that infection with the ET-12 epidemic clone was the most prevalent strain amongst B. cenocepacia- infected patients. It was also found that pre-transplant strains remained responsible for post-transplant infections. Phenotypic methods for the identification of Bcc strains and closely related organisms have been difficult to develop. A collection of 493 strains including Burkholderia cepacia (genomovars I- IX), Pseudomonas aeruginosa, and other closely related organisms, were investigated for their abilities to produce a wide range of peptidases, glycosidases, esterases and other miscellaneous enzymes using both chromogenic and fluorogenic substrates. The 312 Bcc strains within the collection were also screened for their capacity to oxidise a number of carbohydrates. The heterogeneous nature of all nine Bcc species was confirmed by this study, as was the close phenotypic relationship of B. cepacia and B. cenocepacia. Some substrates, however, were shown to have some taxonomic utility for the differentiation of species within the Bcc and also for closely related organisms. Metabolic activities that showed diagnostic potential included production of 13-ribosidase, 13-xylosidase and 13-glucosidase, as well as oxidation of cellobiose, maltose and trehalose. Screening for palmitate esterase and a or 13-trypsin production was useful for the differentiation of Pandoraea sp. and Ralstonia picketti respectively. CF infections caused by P. aeruginosa and Bcc strains, are most successfully treated using two or three drug combinations. A number of cell wall-acting antibiotics were tested in combination with the phosphonopeptide alafosfalin for synergistic effects against Bcc and P. aeruginosa strains. Alafosfalin was most effective in combination with ceftazidime against Bcc strains, and in combination with tobramycin and ceftazidime as a triple combination against P. aeruginosa.

This study investigated the history of traditional Korean papermaking within its historical context: the relationship with the development of papermaking techniques in neighbouring countries were examined though primary focus was given to the development of materials and tools used. In order to understand the characteristics of historical Korean paper and the development of tools and materials used over time, surveys on Korean and Japanese collections at the British Library and a private Korean collection were carried out. Korean objects dated between the 12th and the 18th century were examined. The data collected from the surveys was compiled in a database and analysed. The data analysis revealed that the thickness of paper was closely related to the thickness of bamboo splints used in manufacture of papermaking screens. Research also included a summary of morphological characteristics and photomicrographs of fibres from nine indigenous plants which were used for traditional Korean papermaking. These standard fibre samples were used as reference to identify the fibres of unknown paper objects surveyed. This fibre identification confirmed the main material to be paper mulberry and, additionally, provided information on supplementary materials including rice straw, reed, hemp, and mechanical wood pulp of coniferous origin — a material that has not been recognised as one of the common supplementary materials in previous studies. In order to provide a better understanding of the materials and tools used in traditional papermaking in Korea, three papermaking experiments were carried out. Firstly, a papermaking experiment was conducted using a mucilaginous substance derived from the roots of Hibiscus Manihot, which has been employed as a formation aid for considerable time in Korea and Japan. Paper samples were then analysed to investigate the physical influence of the substance on the sample sheets. Secondly, a fixed laid screen was designed and sheets were produced using it. The intention here was to support a hypothesis which was proposed by the author in order to explain a possible chronological development of papermaking mould structure in China and its potential spread to neighbouring countries. The last experiment was conducted to simulate a technique of papermaking with reclaimed paper. Although the use of reclaimed paper was recorded in early literature, details of the process were unknown.