Demonstration of a multi-technique approach to assess glacial microbial populations in the field

The ability to perform microbial detection and characterization in-field at extreme environments, rather than on returned samples, has the potential to improve the efficiency, relevance and quantity of data from field campaigns. To date, few examples of this approach have been reported. Therefore, we demonstrate that the approach is feasible in subglacial environments by deploying four techniques for microbial detection: real-time polymerase chain reaction; microscopic fluorescence cell counts, adenosine triphosphate bioluminescence assay and recombinant Factor C assay (to detect lipopolysaccharide). Each technique was applied to 12 subglacial ice samples, 12 meltwater samples and two snow samples from Engabreen, Northern Norway. Using this multi-technique approach, the detected biomarker levels were as expected, being highest in debris-rich subglacial ice, moderate in glacial meltwater and low in clean ice (debris-poor) and snow. Principal component analysis was applied to the resulting dataset and could be performed in-field to rapidly aid the allocation of resources for further sample analysis. We anticipate that in-field data collection will allow for multiple rounds of sampling, analysis, interpretation and refinement within a single field campaign, resulting in the collection of larger and more appropriate datasets, ultimately with more efficient science return

Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction

Acknowledgments The authors would like to thank the Scottish Universities Life Sciences Alliance (SULSA) for their support.Peer reviewedPublisher PD

Remembering Another Aspect of Forgetting

Although forgetting is most often thought of in terms of declines in performance (response loss or impairment), another class of memory phenomena, the forgetting of stimulus attributes, has begun to attract experimental attention. In non-human animals, the loss of memory for stimulus features is reflected in the flattening of stimulus generalization gradients as well as in the attenuation of the disrupting effect of a shift in context at testing. In both cases, a delay between the learning episode and testing results in increased responding in the presence of previously ineffective stimuli. Thus, previously discriminable cues become more functionally interchangeable. The implications of the forgetting of attributes for some theoretical issues of memory loss and for methodological strategies have been noted earlier. However, relatively little is known about the neurobiological mechanisms underlying stimulus attribute forgetting, and why some memories are maintained while others are not. In this paper we review the evidence for the forgetting of stimulus attributes, discuss recent findings identifying neurobiological underpinnings of forgetting and generalization of fear responses, and discuss relevant clinical implications of fear generalization

mini spindles: A Gene Encoding a Conserved Microtubule-Associated Protein Required for the Integrity of the Mitotic Spindle in Drosophila

We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle

Optical modulation of high-affinity biomolecules function via photochromic dyes : a step towards an artificial control of biological activity

Prior to this study, there has yet to be a clear demonstration of an artificial control of antibody affinity via photochromic dyes. The research described in this thesis sets out to address this by investigating photochromic dyes and their subsequent applications with high affinity biomolecules - primarily to photomodulate the functions of biomolecules. The main avenue of investigation explored the conjugation of photochromic dyes (spiropyrans) to proteins (an enzyme and five different antibodies), to achieve reversible photomodulation of protein function for possible applications in biosensor technology (such as the development of reagentless bio¬reversible sensing systems). A secondary aim involved the investigation of the feasibility of antibody-antigen binding in the presence of ionic liquids. Ionic liquids have recently experienced growing interest as replacements for traditional organic solvents in a number of industrial applications. The practicability of spiropyrans in ionic liquids was also investigated (with the future possibility of photomodulated antibody-antigen interactions in ionic liquids to deliver a variety of improved analytical performances). The synthesis and photoswitching properties of an appropriate range of spiropyran dyes are reported. The spiropyran dyes are synthesised to possess a carboxyl group to aid carbodiimide mediated conjugation to lysine amino groups of proteins. The photochromic behaviour of the spiropyran dyes in various solvents, temperature and pH ranges were observed. Conjugation of carboxylated spiropyran dyes to an enzyme: horseradish peroxidise, was initially observed to aid development of experimental protocol for the target study group i.e. antibodies. Photomodulation of the modified horseradish peroxidase was found to demonstrate ~ 60 % decline in enzyme activity, an effect which was reversible as a result of the photoswitching capabilities of the attached spiropyran dyes. The five different antibodies; anti Atrazine, anti GroEL, anti Phytanic Acid, anti FITC and anti Staphylococcus aureus were modified with spiropyran dyes as with horseradish peroxidase. Reversible antibody affinity photomodulation was observed via their reaction in an ELISA which yielded a decline of ~ 15 %, ~ 40 %, ~ 50 %, ~ 55 % and ~ 65 % in binding signal respectively. A fatigue assessment was conducted on the photoswitching capabilities of both the conjugated and the unconjugated spiropyran dyes. This was expressed as ten photoswitching cycle experiments, the first evenly spaced over ten days and a second over ten weeks. The initial results suggested dye degradation increased with consecutive photoswitching cycles of the conjugated spiropyran dyes. It was observed that the level of degradation for the unconjugated spiropyran dyes was independent to the timing interval between photoswitching cycles, suggesting storage stability of the compound. However the level of degradation for the conjugated spiropyran dyes was dependent to the timing interval between photoswitching cycles, suggesting storage instability. A subsequent study involved the demonstration of the feasibility of antibody-antigen binding in ionic liquids for the first time. Various combination ratios of ionic liquids with aqueous phosphate buffered saline were employed. Initial experimentation of antibody-antigen binding showed that use of solutions with an ionic liquid content of 50 % and below, produced identical results to that of the standard aqueous phosphate buffered saline. At 95 % ionic liquid content, a lower level of binding activity was observed. The possibility of a photomodulated antibody-antigen interactions in ionic liquids did not produce a significant result on this occasion with the observation of spiropyran dyes failure to photoswitch in solutions with as low as 10 % ionic liquid content. In summary, although the development of a reagentless bio-reversible sensing system continues beyond the period of this PhD thesis, significant progress has been made with regards to photochromic antibodies as possible candidates for further studies and applications, also the establishment of antibody-antigen binding in various ionic liquids can serve as ways to further enhance the applicability of such reactions under different environmental conditions.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Optical modulation of high-affinity biomolecules function via photochromic dyes : a step towards an artificial control of biological activity

Prior to this study, there has yet to be a clear demonstration of an artificial control of antibody affinity via photochromic dyes. The research described in this thesis sets out to address this by investigating photochromic dyes and their subsequent applications with high affinity biomolecules - primarily to photomodulate the functions of biomolecules. The main avenue of investigation explored the conjugation of photochromic dyes (spiropyrans) to proteins (an enzyme and five different antibodies), to achieve reversible photomodulation of protein function for possible applications in biosensor technology (such as the development of reagentless bio¬reversible sensing systems). A secondary aim involved the investigation of the feasibility of antibody-antigen binding in the presence of ionic liquids. Ionic liquids have recently experienced growing interest as replacements for traditional organic solvents in a number of industrial applications. The practicability of spiropyrans in ionic liquids was also investigated (with the future possibility of photomodulated antibody-antigen interactions in ionic liquids to deliver a variety of improved analytical performances). The synthesis and photoswitching properties of an appropriate range of spiropyran dyes are reported. The spiropyran dyes are synthesised to possess a carboxyl group to aid carbodiimide mediated conjugation to lysine amino groups of proteins. The photochromic behaviour of the spiropyran dyes in various solvents, temperature and pH ranges were observed. Conjugation of carboxylated spiropyran dyes to an enzyme: horseradish peroxidise, was initially observed to aid development of experimental protocol for the target study group i.e. antibodies. Photomodulation of the modified horseradish peroxidase was found to demonstrate ~ 60 % decline in enzyme activity, an effect which was reversible as a result of the photoswitching capabilities of the attached spiropyran dyes. The five different antibodies; anti Atrazine, anti GroEL, anti Phytanic Acid, anti FITC and anti Staphylococcus aureus were modified with spiropyran dyes as with horseradish peroxidase. Reversible antibody affinity photomodulation was observed via their reaction in an ELISA which yielded a decline of ~ 15 %, ~ 40 %, ~ 50 %, ~ 55 % and ~ 65 % in binding signal respectively. A fatigue assessment was conducted on the photoswitching capabilities of both the conjugated and the unconjugated spiropyran dyes. This was expressed as ten photoswitching cycle experiments, the first evenly spaced over ten days and a second over ten weeks. The initial results suggested dye degradation increased with consecutive photoswitching cycles of the conjugated spiropyran dyes. It was observed that the level of degradation for the unconjugated spiropyran dyes was independent to the timing interval between photoswitching cycles, suggesting storage stability of the compound. However the level of degradation for the conjugated spiropyran dyes was dependent to the timing interval between photoswitching cycles, suggesting storage instability. A subsequent study involved the demonstration of the feasibility of antibody-antigen binding in ionic liquids for the first time. Various combination ratios of ionic liquids with aqueous phosphate buffered saline were employed. Initial experimentation of antibody-antigen binding showed that use of solutions with an ionic liquid content of 50 % and below, produced identical results to that of the standard aqueous phosphate buffered saline. At 95 % ionic liquid content, a lower level of binding activity was observed. The possibility of a photomodulated antibody-antigen interactions in ionic liquids did not produce a significant result on this occasion with the observation of spiropyran dyes failure to photoswitch in solutions with as low as 10 % ionic liquid content. In summary, although the development of a reagentless bio-reversible sensing system continues beyond the period of this PhD thesis, significant progress has been made with regards to photochromic antibodies as possible candidates for further studies and applications, also the establishment of antibody-antigen binding in various ionic liquids can serve as ways to further enhance the applicability of such reactions under different environmental conditions.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Towards a remote portable bio-affinity surface plasmon resonance analyser for environmental steroidal-pollutants

The widespread presence of chemicals with the capacity to disrupt the endocrine system in both wildlife and humans in our natural environment has increasingly become of major concern in the last ten years. Endocrine disrupting compounds (EDCs) are a group of compounds that pose a potentially dangerous and real threat to the health of both humans and wildlife. These substances can mimic or interfere with the biological pathways of natural endogenous signalling chemicals controlling the endocrine system (e.g. sex hormones). Endocrine disrupters are ubiquitous in water. The detection, monitoring and treatment of wastewaters and surface waters for EDCs would significantly help minimise the environmental burden imposed by these natural and synthetic compounds. To optimise such processes, an economical, in-situ or field-based detection technique for EDCs is required. The research presented in this thesis describes the development of a portable surface plasmon resonance device for the detection of endocrine disrupters in wastewater and surface waters. The first two result chapters describe the construction, development and optimisation of the portable analyser and immunoassay protocol using anti-estrogenic antibodies. A novel approach for regenerating the SPR sensing surface was achieved by using Persil biological laundry liquid (1%). The developed immunoassay showed a working range between 0.2 - 7µg/L for Estrone-3-Gulcuronide (E13G) in buffer. The detection of 17beta- Estradiol (E2) in buffer, synthetic wastewater and real wastewater samples was also carried out; the working range was 0.1 - 10µg/L; 0.3-7µg/L and 0.1-10µg/L respectively. The second part of the thesis describes the synthesis and protocol development of a photo-chromic dye and its application to immuno-sensing systems en route to a reversible bio-affinity antibody for application to regenerating biosensing surfaces. This approach was to demonstrate the concept of remote regeneration of the active sensing surface for a portable optical sensor.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of a fluidic sensor for the detection of herbicides using thylakoid preparations immobilised on magnetic beads to aid regenerability

Following the industrial revolution and advances in chemical science, the pollution of the environment with trace organic pollutants has been steadily increasing, which is of concern, due to their effect on the environmental and human health. Tighter legislation that has been introduced in order to minimise the release of harmful pollutants has led to the initiation of monitoring programmes. For example, drinking water suppliers are obliged to systematically monitor drinking water supplied for human consumption for a large range of pollutants. The same applies for waste water treatment facilities. The well-established standard methods of environmental waters analysis require sampling and transportation of samples to the laboratory for detailed measurements. Therefore, the timescale from sampling to reporting is not ideal, as a considerable lag occurs. There is therefore the potential for the use of in situ methods that overcome this issue. As these do not currently exist, a need to address this is identified. Biosensors are sensing devices that rely on a biologically-derived component as an integral part of their detection mechanism. Biosensors that respond to pollutants could be used for rapid, low cost, field-based pre-screening of water samples. Herbicides are considered to be the most important class of pesticides used in the E.U. Herbicides can be highly toxic for human and animal health, and increase in the application of herbicides in agriculture during recent decades has resulted in immense pollution of both soil and water. About half of the herbicides used at present in agriculture inhibit the light reactions in photosynthesis, mostly by targeting the Photosystem II (PSII) complex. A method of detecting certain classes of herbicides is therefore proposed; the photosynthesis-inhibiting herbicides act by binding to PS II, a chlorophyll– protein complex which plays a vital role in photosynthesis, located in the thylakoid membrane of algae, cyanobacteria and higher plants. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by the HRP-mediated luminol chemiluminescence reaction. The design and development of a fluidic sensor unit for the detection of such herbicides, based upon their inhibition of the hydrogen peroxide production, will employ the use of superparamagnetic beads in order to address issues of reuse and regenerability. The illumination-dependent production of hydrogen peroxide by isolated thylakoids, and its inhibition by herbicides in a concentration-dependent manner, were achieved and measured with the HRP-mediated chemiluminescence reaction with luminol in a cuvette, batch format, allowing for the detection of herbicides down to 6.0 x 10-09.The integration of the above reactions has been achieved by designing and constructing a fluidic unit that combines the herbicide-dependent production and the detection of hydrogen peroxide in a single fluidic assay by combining all the individual steps in a compact, portable format, with both HRP and thylakoids covalently coupled on superparamagnetic beads. This addresses issues of regenerability, as the beads are introduced, used and discarded following a measurement, controlled only by magnetic and flow forces. Herbicide detection was achieved to a lower LOD of 5.5 x 10-10 M. The concept development, design and construction of the fluidic unit, as well as results of the detection of herbicides with the batch assay method has been published, in a paper by the author (Talanta, 2008, vol. 77, no. 1, pp. 42-47), Considerable progress has therefore been made towards developing a system that would be suitable for automated, field deployment applications for the detection of the most frequently used classes of herbicides; the lower LOD however is not within the stringent legislated maximum permissible limits set for herbicides measured in water, in European waters. An immediate step forward would be to achieve the required lower LOD, with the unit's development into a prototype instrument that can be field deployed being the further goal.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Detecting life on Mars and the life marker chip : antibody assays for detecting organic molecules in liquid extracts of Martian samples

The Life Marker Chip instrument, which has been selected to fly as part of the 2018 ExoMars rover mission payload, aims to detect up to 25 organic molecules in martian rocks and regolith, as markers of extant life, extinct life, meteoritic in-fall and spacecraft contamination. Martian samples will be extracted with a solvent and the resulting liquid extracts will be analysed using multiplexed microarray-format immunoassays. The LMC is under development by an international consortium led by the University of Leicester and the work described within this thesis was carried out at Cranfield University as part of the consortium’s broader program of work preparing the LMC instrument for flight in 2018. Within this thesis four specific areas of LMC instrument development are addressed: the investigation of immunoassay compatible liquid extraction solvents, the study of likely interactions of martian sample matrix with immunoassays, the development of antibodies for the detection of markers of extinct life and demonstration of solvent extraction and immunoassay detection in a flight representative format. Cont/d.EThOS - Electronic Theses Online ServiceGBUnited Kingdo