Purposive variation in recordkeeping in the academic molecular biology laboratory

This thesis presents an investigation into the role played by laboratory records in the disciplinary discourse of academic molecular biology laboratories. The motivation behind this study stems from two areas of concern. Firstly, the laboratory record has received comparatively little attention as a linguistic genre in spite of its central role in the daily work of laboratory scientists. Secondly, laboratory records have become a focus for technologically driven change through the advent of computing systems that aim to support a transition away from the traditional paper-based approach towards electronic recordkeeping. Electronic recordkeeping raises the potential for increased sharing of laboratory records across laboratory communities. However, the uptake of electronic laboratory notebooks has been, and remains, markedly low in academic laboratories. The investigation employs a multi-perspective research framework combining ethnography, genre analysis, and reading protocol analysis in order to evaluate both the organizational practices and linguistic practices at work in laboratory recordkeeping, and to examine these practices from the viewpoints of both producers and consumers of laboratory records. Particular emphasis is placed on assessing variation in the practices used by different scientists when keeping laboratory records, and on assessing the types of articulation work used to achieve mutual intelligibility across laboratory members. The findings of this investigation indicate that the dominant viewpoint held by laboratory staff other than principal investigators conceptualized laboratory records as a personal resource rather than a community archive. Readers other than the original author relied almost exclusively on the recontextualization of selected information from laboratory records into ‘public genres’ such as laboratory talks, research articles, and progress reports as the preferred means of accessing the information held in the records. The consistent use of summarized forms of recording experimental data rendered most laboratory records as both unreliable and of limited usability in the records management sense that they did not form full and accurate descriptions that could support future organizational activities. These findings offer a counterpoint to other studies, notably a number of studies undertaken as part of technology developments for electronic recordkeeping, that report sharing of laboratory records or assume a ‘cyberbolic’ view of laboratory records as a shared resource

Role of the complement factor H-related protein 5 in renal disease by protein expression and molecular solution structural studies

Complement Factor H-Related 5 (CFHR5) belongs to the same complement family as the major regulator Factor H. CFHR5 comprises nine short complement regulator (SCR) domains. The duplication of the N-terminal SCR-1/2 domains causes CFHR5 nephropathy, a cause of kidney failure in Cypriots. To clarify the molecular basis of CFHR5 nephropathy, E. coli expression systems were developed for SCR-1 and SCR-1/2 of CFHR5, and recombinant CFHR5 SCR-1/9 was obtained from a commercial mammalian expression system. First, the domain arrangement of CFHR5 SCR-1/9 was studied by analytical ultracentrifugation and X-ray scattering. Sedimentation velocity reported a molecular mass of 134 kDa, indicating that CFHR5 is dimeric. The CFHR5 sedimentation coefficient of 5-6 S decreased with increased NaCl, showing that this became more extended. X-ray scattering also showed that CFHR5 was dimeric. The X-ray mean radius of gyration RG was 5.5 ± 0.2 nm, and its maximum length was 20 nm. This length is low compared to that of 32 nm for monomeric Factor H with 20 SCR domains, indicating that CFHR5 possessed a more compact SCR arrangement than that of Factor H. Atomistic scattering curve modelling of CFHR5 that involved Monte Carlo simulations to generate physically realistic atomistic SCR structures showed that CFHR5 possessed a folded-back compact domain structure. Second, sedimentation velocity showed that SCR-1 was monomeric, while SCR-1/2 was dimeric, thus locating a CFHR5 dimerization site to its N-terminus. In summary, the solution structure of CFHR5 is markedly more compact than previously thought, and its dimerization site was located to SCR-1/2. The perturbation of SCR-1/2 may have a major role in causing CFHR5 nephropathy

Structural studies of putative general stress and related proteins from Deinococcus radiodurans

This study describes the cloning, expression, purification, biophysical characterisation and crystallisation of DR_1146; a putative general stress protein from the extremophilic bacterium Deinococcus radiodurans (R1). The extraordinary ability of D. radiodurans to resist mutation or apoptosis on exposure to high does of ionising radiation has formed the basis of a structural genomics project underway at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The work presented in this study forms part of the ESRF’s D. radiodurans initiative, and was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the ESRF as an Industrial Cooperative Award in Science and Engineering (CASE) PhD studentship. A period of one-year was spent on secondment at the ESRF, working within the Macromolecular Crystallography Group. Several constructs of the dr_1146 gene have been successfully overexpressed in E. coli cells to give high yields of target protein. Purification by immobilised metal affinity chromatography (IMAC) was facilitated by the incorporation of a 6xHis tag and supplemented by a final gel filtration step. Although high purity levels were achieved, imaging by SDS-PAGE analysis identified that DR_1146 was susceptible to stringent proteolysis. It is thought that initial crystallisation trials were unsuccessful due to inhomogeneity of the sample caused by reported degradation of the target protein. Biophysical characterisation of DR_1146 by isothermal titration calorimetry (ITC) and fluorescence spectroscopy (FS) identified a moderate affinity of 4-11 μM for the flavin molecules, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Differential scanning calorimetry (DSC) and circular dichroism (CD) experiments demonstrated an increase in chemical and thermal stability of the protein on binding to the flavin molecule, FMN. Analytical ultracentrifugation (AUC) and Nuclear magnetic resonance (NMR) spectroscopy were employed to investigate the solution behaviour of DR_1146 in the presence of FMN. AUC results uncovered a monomer-dimer equilibrium; with DR_1146 self-associating to form a dimer at a concentration of 7.67 μM. NMR spectroscopy depicted that global changes occur within the structure of DR_1146 on binding to FMN. The high quality of spectra obtained showed potential for 3-D structure determination by NMR if ordered crystals could not be obtained for X-ray diffraction. Interestingly, analysis of NMR spectra proved to be integral to identifying a homogenous sample for successful crystallisation of DR_1146. By monitoring chemical shifts it was possible to determine the time needed for degradation of DR_1146 to cease, and the amount of FMN needed to ensure saturation of binding sites. From this particular sample, a stable 28 kDa fragment was isolated by gel filtration. Automated sitting-drop vapour-diffusion experiments resulted in the growth of yellow DR_1146-FMN crystals for which, although poor in quality, X-ray diffraction was obtained. Overall this study reflects the importance and advantage of incorporating information gained from biophysical characterisation into the strategies employed for successful protein crystallisation. The characterisation of DR_1146 as a flavoprotein points towards a possible role in electron transfer due to the extensive redox capacity of flavin. This could implicate the protein in the production of damaging reactive oxygen species (ROS) as a result of irradiation, contributing to oxidative stress levels. Alternatively, if DR_1146 is identified as a FMN-binding pyridoxine 5'-phosphate oxidase (PNPOx) enzyme, as sequence homology suggests, it could play a role in detoxification and stress response through production of pyridoxal 5'-phosphate (PLP), a known scavenger of ROS. Only further characterisation and elucidation of a 3-D structure would confirm or dispel these functional hypotheses and ultimately provide a greater understanding of how D. radiodurans is able to deal with such oxidising conditions. Simultaneously, experiments were carried out on other soluble and membrane protein targets from D. radiodurans and their corresponding homologues from Streptococcus pneumoniae (TIGR4). The aim of comparable studies was to identify key structural or functional differences between the two Gram-positive bacterial strains. Identification of features unique to D. radiodurans, but unconserved in S. pneumoniae, could contribute to further understanding of bacterial radioresistance. SP_1651 is a thiol peroxidase which forms part of the Mn-ABC transport system in S. pneumonia. Its homologue from D. radiodurans, DR_2242 is a putative thiol-specific antioxidant protein, the structure of which has been solved by Dr. Dave Hall as part of the ESRF’s structural genomics project (unpublished). The aim of this part of the project was to elucidate the structure of SP_1651 so that a comparison with DR_2242 could be made. The sp_1651 gene (psaD) was successfully expressed and purified to homogeneity by IMAC and gel filtration. After the proteolytic removal of a 6xHis tag, the purified protein was crystallised by sitting-drop vapour-diffusion. Preliminary diffraction with a resolution limit of 3.2 Å was obtained, however data showed high mosaic spread. Unfortunately, attempts to reproduce initial crystals failed and hence, structural comparisons with DR_2242 could not be made. DR_0463 is a 108 kDa maltooligosyltrehalose synthase (MTSase) which has been shown to catalyse the breakdown of maltooligosaccharide (or starch) into the disaccharide, trehalose. The full length gene was expressed in BL21(DE3)pLysS cells, producing large yields of insoluble target protein. DR_0463 was solubilised with 8 M Urea and then purified by IMAC in the presence of the denaturant. The low affinity of DR_0463 for the Ni2+ matrix of the HisTrap column proved to be problematic when trying to obtain homogeneity. However, by sequentially repeating IMAC purification up to three times with the same protein sample, a large proportion of impurities were removed. SP_1648 (PsaB) is an ATP-binding protein that forms part of the Mn-ATP transport system in S. pneumoniae and its homologue from D. radiodurans, DR_2284 is predicted to share similar function. Purification of soluble SP_1648, expressed in B834(DE3) cells, was complicated by an inability to bind the protein to the column matrix for IMAC. In the case of DR_2284, expression trials yielded only a minute amount of insoluble protein in BL21-AI competent cells. The bottlenecks in early expression and purification stages provided valuable experience in dealing with problematic proteins. As an introduction to molecular cloning, two genes predicted to encode integral membrane proteins from D. radiodurans, were cloned for preliminary expression trials. This work was carried out at the ESRF and contributed to an extension of the structural genomics project, to incorporate membrane protein targets from D. radiodurans. Full length forms of the genes thought to encode an undecaprenyl diphosphatase (UDP) and a diacylglycerol kinase (DGKA) were successfully cloned in to pET-28b, with incorporation of separate N- and C- terminal 6xHis tags

Biochemical characterisation of unusual glycolytic enzymes from the human intestinal parasite Blastocystis hominis

Blastocystis is an important parasite that infects humans and a wide range of animals like rats, birds, reptiles, etc. infecting a sum of 60% of world population. It belongs to the Stramenopiles, a Heterologous group that includes for example the Phythophthora infestans the responsible for the Irish potato famine. Previous work had reported the presence of an unusual fusion protein that is composed of two of the main glycolytic enzymes; Triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (TPI-GAPDH). Little is known about this protein. Blastocystis TPI-GAPDH and Blastocystis enolase were both characterized biochemically and biophysically in this project. The phylogenetic relationships of those two proteins among other members of either Stramenopiles, or other members of the kingdom of life were examined and found to be grouping within the chromalveolates. Our studies revealed that those two proteins, Blastocystis enolase and Blastocystis TPI-GAPDH, had a peptide signal targeting them to the mitochondria. This was an unusual finding knowing that text books always referred to the glycolytic pathway as a canonical cytoplasmic pathway. Structural studies had also been conducted to unravel the unknown structure of the fusion protein Blastocystis TPI-GAPDH. X-ray crystallography had been conducted to solve the protein structure and the protein was found to be a tetrameric protein composed of a central tetrameric GAPDH protein flanked with two dimmers of TPI protein. Solving its structure would be the starting point towards reviling the role that TPI-GAPDH might play in Blastocystis and other organisms that it was found in as well. Although a fusion protein, the individual components of the fusion were found to contain all features deemed essential for function for TPI and GAPDH and contain all expected protein motifs for these enzymes

High-throughput screening technologies for identification and expression of functional domains of proteins of biomedical importance

The ability to produce multi-milligram quantities of a recombinant ‘target’ protein in a proteolytically stable, soluble, and functional form is often necessary for subsequent biochemical, biophysical and structure-based analyses. Sub-constructs expressing only part of a large target protein can often be useful. Combinatorial Domain Hunting (CDH) is a methodology that allows the rapid production of sub- constructs via a random DNA fragmentation technique. One particular issue with CDH is that it can be used to identify globular regions or domains of a target protein, but does not take account of the functional properties of such domains; therefore some ‘hits’ are not useful, because they exclude these functional regions. Here, we have attempted to enhance the CDH methodology by including an additional screening step that could specifically identify those constructs expressing functional protein domains. However, whilst rigorous testing of this functionality screen proved it to be successful under selective conditions, it was not considered suitable for inclusion in the CDH method. CDH was also used to identify highly expressed, proteolytically stable regions of a previously largely uncharacterized protein, and to investigate their functionality. Human Claspin is a large, highly charged, S=phase specific ‘molecular scaffold’ protein, with no identifiable sub-domains or enzymatic function(s). However, Claspin is known to make multiple different protein-protein interactions at replication forks during the intertwined processes of DNA replication and DNA replication-coupled repair. CDH successfully identified a number of N-terminal expression constructs that could be expressed and purified to a high degree of homogeneity. Structural and functional analyses of these protein fragments indicated that the N-terminus of human Claspin is intrinsically disordered, and elongated in nature. However, these regions may become ordered upon binding to their respective protein or macromolecular partner(s). Furthermore, several N-terminal fragments were found to be able to bind to both single- or double-stranded DNA when longer than 16 nucleotides/base-pairs in length. Additionally, the phospho-specific protein-protein interaction made by human Claspin, with the checkpoint kinase Chk1 was further investigated

Expression, inhibition and in silico modelling of human neuronal nitric oxide synthase

Nitric oxide (NO) is a free radical, gaseous molecule that is involved in a vast range of biological signalling processes including long term memory formation, neurogenesis, vasodilation and inflammation. The molecule is produced by a family of enzymes called nitric oxide synthases (NOS). There are three major isoforms of these NO producing enzymes, endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). eNOS is constitutively expressed in the circulatory system, while iNOS is inducibly expressed in response to cellular invasion by a pathogen. nNOS, which was the isoform targeted in this work, is constitutively expressed in the brain and central nervous system, where the NO it produces regulates many neurogenic pathways. Over activation of nNOS leads to elevated levels of NO in the brain. This has been implicated in an array of serious physiological conditions including schizophrenia, Alzheimer’s and Parkinson’s disease. Modulation of nNOS over activation therefore represents a potential therapeutic avenue for treatment of a variety of debilitating and life threatening conditions. The field of nNOS inhibition currently focuses almost exclusively on the arginine binding site of the rat nNOS isoform of the enzyme. The arginine binding site in rat nNOS does differ from that of human nNOS, and as such is not an ideal system with which to study human nNOS inhibition. Therefore, this work incorporated computational studies to model and examine the binding sites of human nNOS. These included construction and verification of a human nNOS homology model. This offered the best option for a detailed examination of the protein as no crystal structure of human nNOS was initially available. The model was used to screen for novel and selective human nNOS inhibitors. This structure based approach was complimented by the use of a ligand based design approach. To do this, a pharmacophore was constructed and used for high throughput screening for the identification of novel inhibitory compounds. The pharmacokinetic properties of the hit compounds were considered, as was their potential selectivity for human nNOS over human eNOS and human iNOS. Over 20 million compounds from the Maybridge and Zinc databases were screened with the various computational methods, and 72 were chosen for biological evaluation. This high throughput in silico screening approach has not been widely utilised in the search for human nNOS inhibitors, with only one such study reported. This publication did not attempt to biologically evaluate the potential nNOS inhibitors. A functional assay was required to test the compounds identified from the in silico screens. This necessitated the recombinant expression of human nNOS, iNOS and eNOS proteins. The literature routinely reports rat nNOS, murine iNOS and bovine eNOS to test inhibitory compounds, so the establishment of an all human high throughput assay system represented a major step forward in the field. Bacterial cells were transformed with the DNA sequences for all three human NOS isoforms. The proteins were recombinantly expressed and purified from the resulting E. coli lysate. The procedure for human nNOS expression was optimised, and the co-expression of human nNOS with human calmodulin was achieved. Calmodulin is a vital co-factor for the production of NO from NOS and its co-expression with human nNOS has not previously been reported. The same approach was applied to eNOS and iNOS. The expressed human NOS proteins were then used to establish a functional, high throughput NOS assay using the Griess reaction. Once established, the assay was used to screen the computationally derived hits for human nNOS inhibitory activity. Some hits could be purchased, but many had to be synthesised. The synthesised compounds were loosely classified into three classes, i) piperazine containing compounds, ii) urea containing compounds and iii) modified arginines and thioethers. The high throughput expression/assay system for human nNOS worked well, and although no novel inhibitory compounds were identified, it allowed for the testing of 72 in silico derived compounds. The assay system also allowed for determination of the IC50 of the non-selective NOS inhibitor, L-NNA. This work represents a valuable contribution to the field of nNOS inhibition as it is among the first projects to attempt large scale screening for human nNOS inhibitory compounds. The projects also succeed in the expression of all three human NOS proteins with human calmodulin, and a high throughput expression/assay system was established for human nNOS inhibition. Novel compounds were successfully synthesised, characterised and evaluated. This work provides a useful roadmap for the future development of human nNOS inhibitory compounds, as well as providing an established computational screening methodology and a high throughput functional human nNOS assays/expression system

A study of clinical strains of Pseudomonas Aeruginosa and the investigation of antibiotic resistance mechanisms in the multidrug resistant strain PA13

Thirteen clinical strains of bacteria from two Irish hospitals were identified as Pseudomonas aeruginosa using classical methods, API 20NE and Biolog GN. Their identification was confirmed by 16S rRNA gene sequencing. The antibiotic resistance profiles of the isolates were determined against forty-one antibiotics belonging to eleven distinct classes. All the isolates were resistant to penicillin G, ampicillin, cephalothin, cloxacillin, oxacillin, amoxicillin, cefotaxime, moxalactam, sulphatriad cotrimoxazole, chloramphenicol and tetracycline. All were sensitive to ceftazidime, piperacillin-tazobactam, cefepime, ceftriaxone, meropenem, aztreonam, amikacin, apramycin, butirosin A, lividomycin and colistin sulphate. One of the isolates, PA13, was resistant to a further fourteen antibiotics and was identified as a multidrug resistant strain. A 2.2 kbp PCR product was amplified from P. aeruginosa PA13. When this product was sequenced it was found to contain four open reading frames. BLASTN analysis identified these as being an integrase gene (ORF1), an aminoglycoside acetyltransferase gene, aac(6’)-Ib (ORF2), an oxacillinase gene (ORF3) and a quaternary ammonium compound resistance gene (ORF4). The presence of the integrase gene and the quaternary ammonium compound gene suggested that the genes were on a Class 1 integron. The acetyltransferase aac(6’)-Ib gene contained the mutant type of the enzyme with a leucine substitution by serine at position 119. Two expression vectors were chosen to investigate the novel oxacillinase gene. One was a commercially available vector, pET-28a (Novagen) and the other was an in-house vector, pPC. The gene was successfully cloned into both vectors. Following induction the desired protein was not expressed in either the soluble or insoluble fractions

Guidebook for genetic resources documentation: A self-teaching approach to the understanding, analysis and development of genetic resources documentation

This guidebook comes with Genebank Management System Software User's Guide, a tutorial and reference guide for use with the Genebank Management System Software (GMS). This guidebook will assist users in making their own decision on the analysis, design, implementation and use of both manual and microcomputer-based documentation systems. (Chinese, Spanish and French versions available