2-hydroxy-6 keto nona 2, 4 diene 1,9 dioic acid 5,6 hydrolase (MhpC) is a homodimeric enzyme belonging to the α/β hydrolase fold family. The three catalytic residues comprise Ser110, Asp235 and His263. To study structure and catalytic mechanism of MhpC, Ser110 was replaced by cysteine and aspartic acid. A range of other mutants were also investigated. The S110C mutant enzyme showed 400 fold reduced catalytic efficiency compared with wild type enzyme, implying that it is a crucial residue in catalysis. The crystal structure of MhpC S110C demonstrated a water molecule interleaving between Cys110 and His263. Exposure of mutant enzyme crystals to substrate showed formation of a covalent complex with the product (hydroxypentadienoic acid) bound through C3 probably by a Michael adduct reaction with the Cys110 thiol. The enzyme-product adduct was confirmed by mass spectrometry. Interestingly, the intact enzyme-substrate complex was also observed by mass spectrometry. The S110D mutant enzyme was not active. The crystal structure of MhpC S110D showed that the active site residue His263 was slightly moved, forming a good hydrogen bond with D110, and many residues in the active site region (Trp264, Phe173 and Phe237) were rearranged. The carboxylate group may provide an interesting mimic of a gem-diol transition state. Serine 40 is close to the catalytic triad and S40C enzyme was 18 fold less active than wild type enzyme. The crystal structure of MhpC S40C revealed an interleaving water molecule between Ser110 and His263. The S40C/S110C double mutant succeeded to form a disulphide bridge redox-switch in the enzyme active site. To disrupt half-of-sites reactivity of MhpC a number of residues were mutated at the subunit interface of the dimeric enzyme. Radical changes in residue type led to mis-folding of MhpC. The more conservative N109G/H256N double mutant enzyme was correctly folded and dimeric although a charged substituent was no longer present in the hydrogen bonding network at the interface. Titration of charged interface residues showed monomeric MhpC by gel filtration at low pH but enzyme activity is low in these conditions.

The majority of people can expect to experience neck pain in their lifetime and some will go on to develop prolonged or repetitive episodes of neck pain or related symptoms. These persistent complaints have become a major cause of disability around the world. Although chronic ‘mechanical’ neck pain can be defined in clinical terms, the underlying pathology remains unclear. Research has failed to demonstrate a consistent relationship between the presence of neck pain and pathology such as degenerative changes. As such, there has been an increasing interest in altered neuro-muscular-articular function in the pathogenesis of neck pain. Over the last 17 years, the role of cervical proprioceptive (mechanoreceptive) dysfunction in the perpetuation of chronic neck pain has received increasing attention from researchers and clinicians. This is commonly referred to as cervicocephalic kinaesthetic sensibility (KS). Cervicocephalic KS has generally been studied utilising head repositioning accuracy (HRA) tasks. At the beginning of this study only seven reports had been published in the area. Following review of this literature, several focused areas of interest were apparent for further study - 1) comparison of KS in various patient subgroups (e.g., insidious onset vs ‘whiplash’); 2) development of more sophisticated methods of measuring head movement and repositioning errors; 3) establishing the characteristics of the tests such as method agreement and reliability; 4) comparison of the discriminative value of repositioning tasks to both subjective ‘straight ahead’ (SSA) and to non-neutral set points within the cervical range of motion (non-straight ahead or nSA). Investigation 1 used a laser pointer method to study head repositioning errors in patients with chronic neck pain of insidious onset. The results suggested that these patients with chronic neck pain show little evidence of impaired cervicocephalic KS, when measured as HRA-SSA. The study also served to highlight several difficulties with the laser pointer method of measurement and the relatively poor knowledge of HRA in healthy subjects. Most previous studies used the mean of 10 repetitions for the measurement but more recent studies utilised fewer repetitions. Although the laser pointer method is simple, inexpensive and easy to use, the method involves a degree of experimenter bias and inaccuracy. It also does not lend itself to concurrent evaluation of variables such as range of motion and speed of head movement. Investigations 2 & 3 focused on the development of the testing method and the introduction of the Zebris CMS 70P ultrasound system for the recording of HRA-SSA, namely; method agreement between a 5 and 10 repetition measuring protocol; method agreement between the laser pointer and Zebris system; the intra/inter-examiner reliability of measurement methods. The Zebris system results suggested that the two methods of measurement agree sufficiently well for the 5 repetition method to replace the 10 repetition method to obtain a mean HRA score and that both could be used interchangeably. Further results suggested that the Zebris and laser pointer methods do not agree sufficiently well to be used interchangeably. The test-retest reliability was comparable between both methods suggesting that from this perspective, either could be used for measuring HRA-SSA. The inter-rater test-retest reliability was comparable to the test-retest reliability suggesting that trained examiners could be interchanged when carrying out repeated measurements. Investigation 4, the final study in this thesis, investigated HRA with the Zebris system using the 5 repetition protocol in two groups of chronic neck pain patients; insidious onset and neck pain from a ‘whiplash’ injury. The results suggested that patients with chronic neck pain of both insidious onset and from a ‘whiplash’ injury show little evidence of impaired cervicocephalic KS when measured using HRASSA and nSA tests. These results conflict with previous studies Despite numerous investigations over the last 17 years, a test that can be routinely applied in the clinical setting for the purposes of diagnosis and treatment monitoring has not been established. Studies to date suggest that an active HRA test to SSA/NHP that is established by the patient may have the greatest discriminative value. Although HRA testing shows some promise in identifying deficits in ‘whiplash’ patients, it is unlikely that the KS tests identify specific subgroups of chronic neck pain patients. There have been several contradictory studies which have shown considerable overlap between patient and healthy groups. It is also unlikely that HRA tests represent a unique test of cervical proprioceptive function (peripheral or central integration) and therefore provide a test exclusive to neck disorders.

MtDNA sequences and microsatellite loci were used as independent molecular markers to reveal the genetic population structure in three deep-sea demersal fish species from the North Atlantic. In the Azores archipelago, the demersal fishery is the second most important after tuna and Helicolenus dactylopterus, Beryx splendens and Beryx decadactylus are among the most captured species. Partial sequences for the mitochondrial control region and cytochrome b gene were obtained for the three species using universal primers. The diversity encountered for these markers is consistent with diversity found on other marine fishes, except for B.decadactylus in which the d-loop and cyt b diversity was lower than expected. Haplotype data indicated a strong genetic differentiation between Helicolenus dactylopterus NW, Cape Verde and NE Atlantic populations suggesting long distance colonisation processes by jump dispersal events along major oceanic currents. Eight microsatellite loci were developed for H.dactylopterus in order to resolve population structure at a finer intraregional scale (within Portuguese waters). Significant deviations from allelic frequencies expected under Hardy-Weinberg equilibrium were detected at several loci. Analysis of FIS revealed significant differences from zero as a result of heterozygote deficiency. Estimates of FST, RST and AMOVA were also significant, suggesting that the population structure of this species within Portuguese waters was not homogeneous. Pairwise comparisons of FST, RST and genetic distances (DSW and (s)2) between populations revealed a significant separation of the Azores and Peniche (continental Portugal) populations as well as a moderate differentiation among subpopulations of the Azores archipelago. Beryx splendens and Beryx decadactylus are two congeneric species with many similarities in known biology. Analysis of haplotype data revealed striking differences in structure and history of the populations of these two species. MtDNA sequences confirmed that Beryx splendens is constituted by one panmitic population within the Northeast Atlantic as has been previously hypothesised by other authors. Surprisingly, indices of genetic diversity were lower in the closely-related B.decadactylus and there was a strong genetic differentiation between Cape Verde and the rest of the NE Atlantic populations when analysed for the same molecular markers. Differences found are discussed based on the limited knowledge of these species especially with respect to lifehistory. Population structure results are discussed in relation to historical and on-going hydrogeographic events. Evidence for the strong influence of several events previous to the last glacial maximum (LGM) on the population demographic history and evolution of deep-sea demersal fish species in the North Atlantic was found.

The nearshore Antarctic marine environment is unique, characterised by low but constant temperatures that contrast with an intense peak in productivity. As a result of this stenothermal environment, energy input has a profound ecological effect. These conditions have developed over several millions of years and have resulted in an animal physiology that is highly stenothermal and sometimes closely coupled with the seasonal food supply, e.g. reproductive periodicity and food storage. Therefore, Antarctic marine animals are likely to be amongst the most vulnerable species worldwide to environmental modifications and can be regarded as highly sensitive barometers for change. Reproductive success is a vital characteristic in species survival and evaluation of change in reproductive condition with time key to identifying vulnerable taxa. Characterising reproductive success with time is a major requirement in predicting species response to change and the early stages of species loss. Some invertebrates are highly abundant in shallow water sites around the Antarctic and form conspicuous members of the Antarctic benthos. Three common echinoderms and one nemertean were sampled from sites adjacent to the British Antarctic Survey’s Rothera Research Station, Adelaide Island, on the West Antarctic Peninsula between 1997-2001. Reproductive patterns were determined by histological analyses of gonad tissue. This study provided further evidence for inter-annual variation in Antarctic gametogenic development, which appeared to be driven to some extent by trophic position and reliance on the seasonal phytoplankton bloom. The largest variation in reproductive condition was demonstrated for the detritivorous brittle star, Ophionotus victoriae. The seasonal tempos of this echinoderm have been attributed in part, to the seasonal sedimentation events common in the high Antarctic. The reproductive patterns in the scavenging starfish, Odontaster validus and the predatory nemertean, Parborlasia corrugatus showed less inter-annual variation. The de-coupling of these invertebrates from the intensely seasonal phytoplankton bloom appeared to partially account for the reproductive trends observed. The lack of inter-annual variation in the reproduction of the filter-feeding sea-cucumber, Heterocucumis steineni, was somewhat counterintuitive, although problems with sample processing probably accounted for the majority of this anomaly. Echinoderms were also collected during the Antarctic summer field seasons in 2003 and 2004. A series of fertilisation success studies were undertaken comparing the adaptations in an Antarctic and an equivalent temperate starfish to achieve optimal numbers of fertilised eggs, and elemental analyses were used to estimate the nutritional and energetic condition of the bodily and reproductive tissues in two Antarctic echinoderms. Fertilisation studies indicated that Antarctic invertebrates require 1-2 orders of magnitude more sperm to ensure optimal fertilisation success. These sperm tended to be long-lived and capable of fertilising eggs 24+ hours after release. The study suggested that synchronous spawning, aggregations and specific pre-spawning behaviour are employed to help counter the deleterious effects of sperm limitation. The Antarctic eggs and sperm were also highly sensitive to even small modifications in temperature and salinity, affecting the number of eggs fertilised. Such stenothermy is of particular relevance if the 1-2ºC rise in global temperature, predicted over the next century, is realised. Biochemical composition of body components of two species of Antarctic echinoderm indicated a significant difference in the composition between the male and female gonad, particularly in the Antarctic brittle star Ophionotus victoriae. The ovaries contained a much larger proportion of lipid compared to the testes, and demonstrated a distinct seasonality in composition. Higher levels of lipid were observed in the ovary during the austral winter coincident with a period of reproductive investment and maturing oocytes in the gonad. O. victoriae exhibited lower amounts of lipid in the late austral spring suggesting the removal of mature oocytes from the ovary through spawning. The seasonality in composition and the high levels of lipid and protein measured in the ophiuroid gut tissue, suggested the gut might play a role in providing material and energy for metabolic function and possibly gametogenesis; higher lipid levels were apparent during the period of seasonal phytodetrital flux. The role of the pyloric ceaca in asteroids as a nutrient storage organ was also evident in the high levels of both protein and lipid observed in this bodily component in the star fish, Odontaster validus.

Stem cell based tissue engineering is viewed as a promising approach for orthopaedic reparative medicine and the application of microfluidic techniques for isolation and characterisation of individual skeletal stem cells is considered a potential source of cells for regenerative medicine. The studies described in this thesis aim to develop original techniques for isolation and characterisation of mesenchymal stem cells and to examine their possible uses in skeletal tissue engineering. These studies developed novel microfluidic technology using dielectrophoretic ring traps and sorting gates for isolation and recovery of specific cells according to immunofluorescent intensity. To date, the devices outlined in this work are limited by the small number of cells that can be isolated, but are capable of recovering established and primary cell populations with 100% purity for specific markers such as STRO-1, while also displaying potential for on-chip analysis and culture due to the ability to precisely control the device's microenvironment. This study has also investigated 28 day organotypic culture of 3D fetal femur-derived cell pellets at an air-liquid interface. It was demonstrated that addition of serum, ascorbate, dexamethasone and BMP-2 resulted in mimicry of in vivo femur development, while addition of ascorbate and TGF- phenotype, thus offering potential models for both cartilage and early bone development. Analysis of pellets demonstrated that significant pellet diameter at day 1 (greater than 0.8mm) is crucial for maintaining reproducible results in osteogenic and chondrogenic conditions. Furthermore, addition of BMP-2 to fetal femur-derived cells cultured in chemically defined media induced formation of a novel cobblestone cell morphology. Characterisation of the cobblestone cells demonstrated a primitive adipogenic phenotype as indicated by the lack of endothelial and haematopoietic marker expression including CD146, TIE2, CD34, and CD105 and upregulation of mesenchymal differ lipid. Overall these studies have offered a novel approach to stem cell isolation for characterisation and have furthered the knowledge of fetal femur-derived cell and their potential as an alternative cell source for skeletal tissue engineering.

Toxicity of dissolved copper was examined in a common coastal mysid population {Praunusflexuosus). The life cycle and ecophysiology were studied under natural conditions throughout the year, and responses to dissolved copper were determined in the laboratory. Pronounced and ontogenic seasonal differences in copper toxicity for the mysid Praunus flexuosus were observed. Sublethal and lethal parameters (mortality, behaviour, metabolism, reproduction, and bioaccumulation) showed seasonal variation in response to copper toxicity, being highest toxicity in summer. Changes in the form of dissolved copper were measured during toxicity testing, using the chelating resin method to provide a measurement oftotal and labile (Chelex-available) dissolved copper in the natural seawater used in the toxicity tests. Labile dissolved copper did not show significant variations in the test seawater, confirming that organisms were exposed to constant labile copper concentrations during the toxicity test. The total dissolved copper concentration was significantly higher than the labile form, as organic complexation occurred in natural seawater and during toxicity tests. The labile fraction could be less than 50% ofthe total fraction. The total dissolved copper concentration decreased significantly when the organism was under stress. While the mortality of the population was insignificant after 10 days of copper exposure (0, 5,25, 75 and 200g 1"') in winter, lethal effects occurred at every copper exposure level after 24 hours (96h LC50 =30.8 g 1"') in summer. The effects of copper on metabolism (respiration and excretion) were very sensitive indicators of sublethal toxicity, which resulted in lethal effects with a prolonged time of exposure. Metabolism shifted to a greater reliance on protein catabolism under copper exposure in both seasons, demonstrating a stronger effect in summer. Total copper content accumulated in the organism increased with increasing copper concentration in solution. Copper accumulation rate was higher in summer . than in winter, increasing to rates of7^g g*1 dry weight day"1. Reproductive processes were severely disrupted at any copper treatment. Production ofjuveniles was reduced to zero, because of the high abortion rate, reduction on brood survival and damage to fertilisation processes.

Phytoplankton exert a dominant influence on the biogeochemical cycling of the oceans, but iron limitation in this dynamic environment can exert a control on photosynthesis. Phytoplankton evolved coping mechanisms to overcome and alleviate the effects of iron limitation. One mechanism is the alteration of the thylakoid membrane and the expression of chlorophyll-binding proteins, which can alter the variable chlorophyll fluorescence signal. Firstly, a study of the chlorophyll-binding iron-stress induced protein, IsiA, in Synechocystis PCC 6803 revealed a 60% increase under iron limitation, in agreement with the theoretical increase. On progressive iron-stress IsiA continued to accumulate without a concomitant increase in _PSI, while Fv/Fm, a measure of photochemical efficiency, continued to decrease. Secondly an oceanographic study to the high latitude North Atlantic in which chlorophyll fluorescence kinetics were used to measure the response to iron addition of in situ phytoplankton populations. The difference in the Fv/Fm between nutrient amended and control treatments (_(Fv/Fm)) was used as a measure of the relative degree of iron stress. The combined observations of both longterm (> 24 h) and short-term (24 h) indicated variability in the seasonal cycle of iron stress, with phytoplankton iron stress developing during the transition from prebloom to peak bloom conditions. Thirdly, similar physiological characteristics were also observed in an oceanographic study in the Ross Sea. The results further confirmed the highly variable response across temporal and spatial scales, but also within different phytoplankton groups. Consistent across all three studies is the reduction in Fv/Fm as the result of an elevated Fo signal, representing potentially unbound chlorophyll-binding proteins. These unbound chorophyll-binding proteins can dominate the total cellular chlorophyll, at least in culture, and reflect a large resource investment. These proteins may provide a rapid source of chlorophyll upon iron resupply. Irrespective of the underlying causes of unbound chlorophyll-binding proteins, the potential large scale expression of such complexes provides a powerful diagnostic tool with which to investigate iron stress in situ.

“Live” (stained) and dead macrofaunal (>300 μm fraction) foraminifera in multicorer samples (0-1 cm and 0-5 cm layers) were analysed at six stations along a transect (100-3400 m water depth) across the Oman margin (Arabian Sea) oxygen minimum zone (OMZ). Very high abundances (2858 per 25.5 cm2), dominated by Uvigerina ex. gr. semiornata, were found in the upper 100 m. The 850 m site also had elevated abundances. These peaks probably represented upper and lower OMZ boundary edge effects, respectively. A total of 199 live species was recognized. Diversity was depressed between 100 m and 850 m and relatively higher at the 1250 m and 3400 m sites. Vertical distribution in the sediment reflected responses found across the horizontal gradient, with species concentrated in the top sediment where bottom-water oxygen concentration was low and distributed more evenly through the sediment where concentration was higher. In general foraminifera and metazoan responded similarly to oxygen and food availability, except that the lower boundary of edge effect was located at a shallower depth (700 m) for the metazoans. Live:dead ratios of foraminifera increased with water depth. The second part of the thesis concerns Gromia, a large marine protist with filose pseudopodia and an organic test that is abundant in the bathyal Arabian Sea. Deep-water Gromia-like morphospecies were discovered in the 1990’s but their relation to shallow-water species was not established. Little is known about gromiid diversity, reflecting the fact that these relatively featureless protists have few characters useful for species identification. Consequently, ultrastructural and molecular techniques were used to examine gromiid diversity on the Oman and Pakistan margins of the Arabian Sea (water depths 1000-2000 m). In total, 27 deep-sea gromiid sequences of the SSU rDNA gene and 6 sequences of the ITS rDNA region were obtained. The data confirmed that Gromia-like protists from the bathyal deep sea are related to shallow-water gromiids. Among Arabian Sea Gromia, seven lineages were identified based on molecular evidence. Five of them form a monophyletic group branching as a sister group to shallow-water species. Four lineages can be defined morphologically, while grape-like morphotypes include 3 lineages that cannot be distinguished morphologically. Each lineage probably represents a separate species, implying that deep-sea gromiid diversity is higher than indicated by their simple morphology. Morphological analysis adds 2 more species, giving a total of 9 deep-sea gromiid species, adding considerably to the number of known marine gromiids, only three of which are currently described.

No description available.

The spawning season of the most commercially important European abalone {Haliotis tuberculata) was evaluated during the summers of 1996,1997 and 1999. The results of the present study show that the best time to start with semi-artificial spawning is from mid July using cultured abalone from the open sea in Guernsey, Channel Isles. Until 1996 Tetraselmis sueccica was the main source of food for juvenile H. tuberculata during their rearing stage (one to twelve weeks old) on glass plates. Nevertheless, this study showed that T. sueccica, as a sole food, is not sufficient for successful development and growth of early juvenile abalone. However, marine diatoms such as Skeletonema costatum, Navicula ramosissima and Cylindrotheca closterium were easily ingested and assimilated depending on the size of the animal and conditions of culture. This thesis studied the effect of different diets (mix of fresh seaweed, fishmeal and an abalone commercial) and temperatures (15, 18 and 22C) on growth and energy budget of juvenile abalone H. tuberculata over a 210 day period. Energy budget was obtained by Ingestion (I), Egestion (E), Somatic growth (Pg), Reproductive investment (Pr), Excretion (U) and Pedal mucus production (M). All these parameters were assessed for grouped and individual organisms. Animals fed on formulated diets (energetically rich) and cultured at 18 and 22C were shown to give better growth rates than the natural diet and preferentially allocated energy to gonad development. Thus, the combination of diet and temperature were factors that contributed to enhance growth rates and gonad development. The abalone commercial diet (CO) used in this study produced shell deformation in 89 % of the population after four months of feeding juvenile abalone. The information of these studies have important implications when considering the nutritional requirements of cultured abalone when fed on formulated diets, which need to contain not only the energy, but also the necessary micronutients which are required to produce good growth rates and also healthy animals.