Recent advances in the area of nanotechnology have led to interesting applications of nanomaterials in medicine, especially in the areas of imaging and treatment. This thesis presents the development of two molecularly imprinted polymers (MIPs) based on the same fluorescent functional monomer. One MIP, prepared in the bulk format, is investigated for its ability to detect tamoxifen and its metabolites. The other MIP synthesised in the nanogel format, holds the potential to be used as pH-responsive drug delivery system. Four objectives were identified within this project. The first was the design and synthesis of fluorescent functional monomer. Two coumarin derivatives carrying a polymerisable unit, for covalent bonding within the polymer, and a carboxylic moiety, for interaction site with the template, were synthesised and characterised. However, only one of them (the VCC: 6-vynilcoumarin-4-carboxylic acid) showed high fluorescent yield and was selected as functional monomer. The second objective involved the development of a detection system based on bulk MIP containing the VCC fluorescent monomer. This system proved effective in generating a detectable signal upon binding the analytes. The signal was observed as a quenching of the polymer fluorescence and it was proportional to the amount of target molecules detected. The third objective was the preparation of tamoxifen-imprinted nanogels for potential application in the drug delivery field. The optimisation of the procedure gave a set of NIP/MIP with the desired solubility, particle size and fluorescence emission. These nanogels were then employed in the last objective, which involved the toxicity study and evaluation of the drug loading on of transgenic line of zebrafish. The nanogels were non-toxic at the tested concentrations and the presence of tamoxifen was confirmed.

Two-component systems (TCS) are the predominant signal transduction pathways in prokaryotes, being present also in eukaryotic organisms, such as algae, fungi and yeast, and higher plants. TCSs play an important role in environmental signal perception and response, essentially implementing adaptation to the surrounding environment. Histidine Kinase 2 (Hik2) in cyanobacteria is a typical sensor histidine kinase, one component of a TCS, and has been identified to be a homologue protein of Arabidopsis Chloroplast Sensor Kinase (CSK). Previous research has elucidated Hik2 to regulate photosynthetic gene transcription with two response regulators, Rre1 and RppA via phosphorylation. A typical histidine kinase contains a variable sensor domain and a conserved kinase domain. It usually functions as a homodimer. This thesis describes the structural characterisation of Hik2, probing particularly its discovered oligomeric states. Results obtained from size exclusion chromatography, native-PAGE, chemical cross-linking analyses and mass spectrometry, amongst others, have shown a variety of Hik2 structural populations exist, further validated by negative stain transmission electron microscopy coupled to single particle analysis. Hik2 protein exists predominantly as a hexamer in low salt conditions, and adding NaCl dissociates hexamers into tetramers, critical for the autophosphorylation activity of Hik2. Thus, a model is proposed for the constitution change of Hik2 oligomers when salt concentration differs. In addition, the sensor domain is typically responsible for detecting environmental input, however, it is not yet clear how Hik2 and CSK sense signals. In this thesis, the structures of Hik2 and CSK sensor domains were analysed and discussed, to aid our understanding of their mechanism of signal perception and transduction.

Controlling crystal polymorphism, the ability of a molecule to crystallise in different solid forms, is one of the grand, ongoing challenges in materials science. In the pharmaceutical industry particularly, where up to half of the active pharmaceutical active ingredients exhibit polymorphic behaviour, it is of paramount importance to rationalise the impact of experimental conditions, such as the nature of the solvent, on the obtainment of a specific c crystal form. As strategies for the selection of polymorphs is still, by and large, based on a trial-and-error approach, it is necessary to acquire a fundamental understanding of the factors controlling the formation of a speci fic solid-state structure during crystallisation from solution. During this doctoral research project, we have conducted a computer simulation study of the early stages of crystallisation of meta-aminobenzoic acid, an important model system in the investigation of polymorphic phenomena. This molecule can in fact form five different polymorphic forms whose selective crystallisation from solution chiefly depends on the nature of the solvent. Molecular models and computational chemistry methods, based on density functional theory and molecular dynamics, have been developed and applied to quantify the processes surrounding the crystallisation of meta-aminobenzoic acid: solvent-solute separation, solute aggregation and surface reactivity. The aim was to identify what controls, at the molecular level, the polymorphic selection process during crystallisation from solution of this important active pharmaceutical ingredient. The results show that the solvent play a signi cant role during the key stages of meta-aminobenzoic acid crystallisation by controlling both the kinetics and thermodynamics of solute desolvation, formation of prenucleation clusters and surface reactivity. This work represents a paradigm of the role of molecular processes during the early stages of nucleation in affecting polymorph selection during crystallisation from solution.

Hypoxia has become a subject of interest among the many environmental stressors as its role in biology is complex and diverse. Hypoxia is a significant low oxygen condition that causes many pathologies and adaptive responses in organisms. It can lead to a moderate or dangerous loss of respiration and can be an indication of tumorigenesis as many tumors lack adequate blood supply. Organisms possess adaptive responses to hypoxia that include hypoxia-inducible factors (HIFs) that activate several downstream pathways that are responsible for altering metabolism and maintaining homeostasis. Within aquatic organisms, hypoxia is an important ecological constraint as oxygen availability within bodies of water can vary greatly over time and space. Therefore, adaptation to hypoxia is likely pervasive, especially in genotypes originating from bodies of water that are prone to hypoxia. Here we report the transcriptional response to acute hypoxia in the clonal freshwater crustacean Daphnia magna. Daphnia were subjected to 1mg/O2 for 12 hours. Then, RNA was extracted, reverse transcribed, and sequenced using Oxford Nanopore MinION. We find that severe hypoxia significantly up-regulates key enzymes in the gluconeogenesis pathway. Additionally, we report genotype-by-environment interactions showing that Daphnia clones from habitats that are hypoxia prone survive better in hypoxia.

Arthrobacter belongs to an underexplored genus of antimicrobial-producing bacteria. All species in this genus are Gram-positive and commonly isolated from soil and marine sources. This genus is known to exhibit metabolic versatility greatly influenced by environmental conditions and nutrition. Arthrobacter sp. strain TAJX1902 isolated from an unknown soil sample is shown to inhibit a filamentous indicator type bacterium. It produced a few bioactive secondary metabolites in rich medium (RM) broth and agar culture. TAJX1902 crude extract was primarily fractionated using a Sephedex LH-20 column. Further purification achieved via Flash Column Chromatography and Preparative Thin layer Chromatography yielded five prominent crude products. The scope of this project is to fully determine the structure of potential novel antibacterial compounds from TAJX1902 via spectroscopic techniques including full 2D-NMR spectroscopic data set in combination with High-Resolution Mass Spectroscopy (HRMS). The research work is currently underway towards the final purification of the compounds using High-Performance Liquid Chromatography (HPLC) method to enable structural determination and evaluation of their antimicrobial activities.

Circadian rhythms are a roughly 24-hour endogenous process that allows organisms to anticipate regular changes in their environment. These rhythms are present in almost all living eukaryotes and regulate important physiological process such as sleep-wake cycles, metabolic changes, hormone release, and activity patterns. In nature, organisms reset their internal clocks to synchronize daily with Earth’s solar day. The inability to synchronize with the environment has shown to result in fitness costs. Spider species within the superfamily Araneoidea have been found to exhibit extreme circadian periods up to five hours different than the 24-hour day, with little evidence of such extremes occurring outside of this superfamily. Studies have also shown that araneoid spiders can undergo large phase shifts without exhibiting any evidence of fitness costs such as reduced survivorship. Araenoid species can re-synchronize their internal clocks to large phase shifts, up to 10 hours daily. The rate of re-synchronization also appears to be accelerated in araneoid species. This indicates a functional difference between the circadian clocks of araneoid species and non-araneoid species. To further investigate differences between the circadian systems of araneoid and non-araneoid spiders, we are conducting a series of phase-shift experiments. Rates of re-entrainment to a six-hour phase shift were compared between an araneoid species (Metazygia wittfeldae) and a non-araneoid species (Pholcus manueli). We hypothesized that the non-araneoid spiders would require a longer amount of time to re-entrain. Results to date are ambiguous. M. wittfeldae, as expected can re-entrain to a six-hour shift within two days. For comparison, such re-entrainment would take a mammal about six days. An interruption in the experimental protocol prevents us from drawing robust conclusions in P. manueli. However, the data suggest that this species may have two circadian oscillators, one which re-entrains to a phase shift rapidly, and one which is highly resistant to re-entrainment.

Oxidative damage to DNA has been implicated in a plethora of pathologies, such as cancer, neurodegenerative diseases, cardiovascular diseases, and aging. One-electron transfer (OET) plays a significant role in oxidative DNA damage in vivo. Guanine as the most oxidizable part of DNA is the major focus of studies on oxidation damage to DNA initiated by OET. Until recently, the pathway of guanine one-electron oxidation via its neutral guanine radical, G·, has been poorly studied. Our recent research has discovered a novel type of products of G· dimerization, D1 and D2, formed as a result of oxidation reaction of guanine derivatives, initiated by OET. A proposed reaction mechanism contains an early intermediate (Int1) generated by recombination of the two G· radicals. We were not able to isolate Int1, so that its role in the proposed reaction mechanism is only hypothetical. Literature data have reported that 8-arylamino-substituted 2´-deoxyguanosine (dGuo) compounds can be oxidized to create structural analogs of D1 and D2. As a result, the original 8-substituted dGuo compounds can serve as analogs of Int1. The goal of this work is therefore to confirm that Int1 is a precursor to D1 and D2 using the analogy approach. Two 8-aryl- and three 8-alkyl-substituted dGuo compounds were synthesized, purified by semipreparative HPLC, and their structures were confirmed by 1H-NMR. 8-subsituted oxidation products analogous to D1 and D2 were obtained from 8-substituted dGuo analogs upon illumination the reaction mixture in the presence of S2O82- as a oxidant and Ru(II)bpy32+ as a photosensitizer at 470 nm. The products were purified by semipreparative HPLC, and their structures were confirmed by 1H-NMR. The purified analogs of D1 were successfully tested for conversion into the D2 analog. Finally, the analogs of D2 were successfully tested for the reaction with primary amines to form analogs of 2-aminoimidozalone (AIz), in agreement with the mechanism characteristic of D2.

Many freshwater ecosystems sustain several invasive species. Here I examine multiple invasions in two highly invaded and well catalogued catchments; Lake Naivasha, Kenya and River Thames, England. New metrics, derived from stable isotope analysis, are used to provide measures of trophic diversity and to examine dietary interactions among species. I test the hypothesis that functionally similar sympatric species will occupy a smaller niche than their allopatric counterparts. Additionally, I quantify the impact of multiple invasive species on ecosystem structure and functioning in order to address the question; do interactions among species amplify or mitigate one another's impact? In Lake Naivasha, the stable isotope metrics revealed serial replacement of invasive species due to dietary interactions. Invasive red swamp crayfish were eventually excluded from the lake due to niche restriction in the presence of a more recent invader, the common carp. Now, the crayfish have migrated into the catchment where they overlap with a species of native river crab. Here, I found a novel mechanism of invasion, whereby the crayfish restricted their niche at the invasion front in order to reduce competition with crabs. Crayfish also caused significant changes in invertebrate community structure and increased decomposition rates, which indirectly resulted in displacement of the crabs. In the Thames catchment, I catalogue the non-indigenous species and show how invasion rates have increased significantly since 1800 due to globalisation. Using the four species of invasive crayfish present (red swamp, signal, Turkish and virile), I demonstrate their extensive diet plasticity using novel measures of niche width and individual specialisation based on stable isotope data. Interactions among the crayfish were examined and this revealed that each species has varying and independent impacts on invertebrate community structure, algal standing stock and decomposition rates. Hence, interactions among invaders are not expected to amplify or mitigate one another's impact and instead, the combined impact will be the sum of their allopatric impacts.

Despite a slow start freshwater meiofauna research is now gathering pace. Evidence is accumulating which indicates the importance of their inclusion in lotic metazoan studies. Here I contribute towards this research effort by conducting an investigation of meiofauna and macrofauna from a chalk stream. I sampled meiofauna for a 19 month period, and macrofauna for a 12 month period between April 2004 and October 2005 from the subsurface, macrophyte stands and gravel beds. The chalk stream community was highly diverse with 57 taxa identified from the subsurface and 186 from the benthos. Meiofauna outnumbered macrofauna in all habitats in terms of density. Both meio- and macroinvertebrates preferred macrophyte stands over gravel beds as habitat, indicated by higher densities, biomass and species richness. Speciesabundance relationships and density-size spectra indicated the invertebrate assemblages of the benthos to be stable over the period of the study as patterns varied little between sampling months and habitats. Production and standing biomass were dominated by the macroinvertebrates which suggests meiofauna had a limited role within functioning of the stream. However, gut content data indicated meiofauna may play an important trophic role, linking basal resources and top consumers. Combined gut content and stable isotope analysis suggested a strong pattern of generalist feeding throughout the whole spectrum of body size in the community, rejecting the concept of functional feeding groups. Predominance of generalist feeding also suggested a large number of weak interactions in food webs. While higher species richness lower in food webs indicated greater functional redundancy of lower trophic levels. Density-body size distributions were shallow with a biased distribution of energy towards larger size classes. Moreover, testing of production, standing biomass and PIB body size allometry was inconclusive with regards to theoretical predictions. The interrelationship ofbiodiversity, stability, and trophic dynamics, with body size determine the structure and dynamics of the chalk stream community, not metabolism.

Saltmarshes in SE England are eroding rapidly and one potential impact is the loss of habitat for fishes. Saltmarshes have been created by setting back the existing line of flood defence through managed realignment. The use by fishes of natural and managed realignment habitats at Tollesbury, Abbotts Hall and Orplands was examined (2005-07). Three seasonal groups were apparent in the fish assemblages of the managed realignment sites: February-April (Pomatoschistus microps and Sprattus sprattus), May-September (Dicentrarchus labrax and Atherina presbyter) and October-January (Liza aurata and Liza ramada). The sites were used mainly by 0- and 1-group fishes and adult P. microps. The mean abundance (July - August 2007) was 558 0.1 ha' (range 76 - 2699 0.1 ha'). In summer, small (< 30 mm) zooplanktivorous D. labrax fed successfully at all sites. Larger (30-59 mm) D. labrax consumed more macroinvertebrates in the Tollesbury managed realignment and two established marshes than at Abbotts Hall and Orplands. By autumn there were no site-specific differences in gut fullness of D. labrax. Stable isotope ratio analysis and gut contents analysis revealed that small (< 50 mm) D. labrax, S. sprattus and A. presbyter assimilated zooplankton which eat detritus, resuspended microphytobenthos and some phytoplankton. L. aurata assimilated zooplankton and microphytobenthos. P. microps (20-50 mm) and A. presbyter (80-99 mm) assimilated benthic meiofauna. Larger (50-230 mm) D. labrax assimilated macroinvertebrates which eat microphytobenthos, Ulva spp., C3 plants and detritus. Some recommendations for saltmarsh restoration are provided with an estimate of the economic value of bass in saltmarshes,to highlight further areas of research.