In mammals, adult neurogenesis in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus produces neurons contribute to learning and memory functions. However, more recent evidence suggests that neurogenesis may also happen in other regions of the brain such as striatum, spinal cord, and hypothalamus (Reviewed in (Riddle, 2007)). It is important to determine if neurogenesis also occurs in non-neurogenic regions of the adult brain such as CB and 3V. This would be of importance for potential future therapeutic applications for brain repair, and also to help understand the fundamental function of the different regions of the adult brain. This thesis tested the hypothesis that neural stem cells (NSCs) are present in the mature cerebellum (CB) and the lining of the third ventricle (3V). Based on this hypothesis, one of the major goals of this thesis was to isolate and characterise NSCs isolated from CB and 3V of adult mouse and weather they could generate neuronal and glial cells in vitro. Immunohistochemical analysis for NSC-associated markers revealed that the mature CB in mouse, chick, and primates contains a population exhibiting NSCs characteristics. Results showed that this population was the Bergmann glial located in the Purkinje cell layer (PCL) of the cerebellar cortex, which express Sox1, Sox2, Sox9, BLBP, and GFAP in a similar pattern. Some of these markers are common for neural stem/progenitor cells or radial glial cells in other brain regions. Observations in the 3V revealed that tanycytes lining the ependymal layer also express NSC and astrocytic markers. Moreover, cells were isolated from the CB and 3V of adult GFP+/-Sox1 mice and tested their ability to form neurospheres, their response to EGF and FGF-2, and their differentiation into neurons, astrocytes, and oligodendrocytes. CB and 3V-isolated cells were found to grow in culture, expand, and differentiate into neuronal and glial phenotypes. It was also observed that CB-derived NSCs could survive and differentiate into neuronal and glial lineages after long term removal of either EGF or FGF-2, although cultures were optimal in the presence of both mitogens. We also observed that cells cultured in either EGF or FGF-2 for 3 weeks had different effects on both CB and LV cells in terms of cell fate specification toward neuronal and glial lineages. This finding suggests the heterogeneity of NSCs population in the adult brain. The identification and mapping of the different NSC populations present in the adult brain offers some important opportunities for regenerative medicine approaches. In order to better characterise the cerebellar population of cells identified above, adult Bergmann glial was observed in a mouse model of cerebellar damage caused by the loss of PCs in pcd5J mutant mice. Calbindin immunostaining at different time-points showed that PC degeneration was visible at P21, then progressed rapidly and became considerable at P26 (nearly 70-80% loss), and by P100 all PCs were lost. Immunohistochemical analysis on sections of CB from pcd and wild type counterparts revealed an increase in Bergmann glia cells at P100 as well as the upregulation of GFAP expression. GFAP+ BG exhibited thick disorganised processes in the molecular layer (ML) at P100 in the mutant mice, with some cell bodies mispositioned in the ML, and significant shrinkage of both ML and internal granular layer (IGL). The increase in the number of Sox1+, Sox2+, and Sox9+ BG in 3 month old mutant mice was not visible at earlier time points analysed. These findings indicate that the PCs loss in pcd5J mice precede and possibly trigger the increase in the Sox1+, Sox2+ and Sox9+ cell population in the CB. Our results also showed that no proliferation activity was observed in the pcd mouse CB at revealed by Ki67 staining, suggesting that the CB microenvironment might not be permissive for neurogenesis even after PCs loss. In vitro isolation of NSCs the CB of P21 pcd mice was carried out, and although cultures appear slower to establish than wild type controls these cells did form neurospheres and express NSC markers. Further characterisation of CB-derived NSCs from pcd mice and their growth and differentiation potential will help better understand the dynamics and possible therapeutic targets for neurodegenerative disorders affecting the CB. The characterisation of CB and 3V derived NSCs from adult mouse CB has provided important information regarding their differences with NSCs derived from neurogenic region in the brain, the lateral ventricle (LV).

Genomic elements such as proteins or genes are the basic unit of the genome and involved in the functioning of every biological process. Predicting, therefore, the function of these genomic elements is the first step in the understanding of functioning of plants under various stress conditions. To date, various types of computational methods have been developed to predict the function of a given protein sequence. The recent increase in the development of a number of methods has created its own set of problems leading to difficulty in applying on newly sequenced genomes especially non-model crops. Due to these reasons, the immediate requirement for development of sophisticated computational methods to predict the function of a given protein sequence is raised. This thesis presents three novel computational tools developed based on transfer learning algorithms to predict the function of a given protein sequence and these tools are: 1) TL-RBPPred, for prediction of RNA-binding proteins, outperformed SPOT-Seq, RNApred, RBPPred and BLASTp on HumanSet (AUC of 0.977), YeastSet (AUC of 0.971), ArabidopsisSet (AUC of 0.972) and GlymaxSet (AUC of 0.97); 2) TL-DBPPred, for prediction of DNA-binding proteins, outperformed DNABP, enDNA-Prot, iDNA-Prot, nDNAProt, iDNA-Prot|Dis, DNAbinder and BLASTp on an testing dataset (AUC of 0.988); and 3) TL-TFPred, for prediction of transcription factors, outperformed PlantTFcat, iTAK and BLASTp on testing dataset (AUC of 0.999) in terms of prediction accuracy. Further, both TL-RBPPred and TL-DBPPred were tested on the transcriptome of the non-model crop, Bambara groundnut (Vigna subterranea (L.) Verdc.), to identify RNA-binding and DNA-binding proteins, respectively. The results obtained from these tests indicated that these two methods outperformed in terms of prediction accuracy (AUC) as compared to existing current state-of-the art tools such as SPOT-Seq, RBPPred, iDNA-Prot and iDNA-Prot|Dis. Based on the performance, the developed methods will be useful in predicting the function of given protein sequences (DNA, RNA-binding and transcription factor) of model species as well as non-model crops.

Apicomplexa parasites such as Plasmodium spp. and Toxoplasma gondii are characterised by a single, lasso-shaped mitochondrion. This organelle is critical during all phases of the parasite life cycle, and is a validated drug target against parasites of the Apicomplexa phylum. In contrast to other eukaryotes, replication of the mitochondrion in these parasites is tightly linked to the cell cycle. A key step in mitochondrial segregation is the fission event, which in many eukaryotes requires the action of Dynamin-related proteins: these mechanochemical enzymes are recruited at the outer membrane of the mitochondria and mediate membrane constriction. To date, none of the components of the apicomplexan fission machinery have been identified and validated. In this work, I investigated the role of DrpC, a highly divergent, Apicomplexa-specific putative Dynamin-related protein, in T. gondii. Endogenous tagging showed that DrpC is adjacent to the mitochondrion, and is localised both at its periphery and at its basal part, where fission is expected to occur. Depletion and dominant negative expression of DrpC results in interconnected mitochondria and ultimately in drastic changes in mitochondrial morphology, leading to parasite death. These data indicate that DrpC is essential for mitochondrial biogenesis in T. gondii. To better understand the fission mechanism, DrpC potential interactors were investigated in this study; intriguingly, it is here shown that the conserved mitochondrial protein Fis1, which in other eukaryotes recruits the Dynamin-related protein during fission, is not required for mitochondrial fission in T. gondii. Conversely, immunoprecipitation data suggest an interaction between DrpC and the microtubule scaffold; as DrpC is also seen at the periphery of the parasite, these data could suggest a second role for this atypical Dynamin-related protein.

This study investigated the effects of heat stress on the survival, mobility, acclimation ability, development, reproduction and feeding behaviour of the brown planthopper Nilaparvata lugens. The critical information derived from the heat tolerance studies indicate that some first instar nymphs become immobilized by heat stress at around 30°C and among the more heat tolerant adult stage, no insects were capable of coordinated movement at 38°C. There was no recovery after entry into heat coma, at temperatures around 38°C for nymphs and 42-43°C for adults. At 41.8° and 42.5oC respectively, approximately 50% of nymphs and adults are killed. In a comparison of the acclimation responses between nymphs and adults reared at 23°C and acclimated at either 15 or 30°C, the data indicate that increases in cold tolerance were greater than heat tolerance, and that acclimation over a generation compared with a single life stage increases tolerance across the thermal spectrum. The temperatures that kill around 50% of nymphs and adults also exert negative effects on development and longevity. The same exposures also lower fecundity and extend egg development time through a combination of mating groups, in which the greatest effects occur when both males and females have experienced sub-lethal heat stress. Likewise, exposure to their ULT50 reduced feeding activity in both life stages of N. lugens. The amount of honeydew excreted by females and males in the treated nymph and adult groups were 3-4x and 2-3x lower than in the equivalent control groups. Overall, sub-lethal heat stress extended egg development time, inhibited nymphal development, lowered fecundity and reduced feeding activity.

The swimming crab Liocarcinus depurator (L.) is a common member of the benthic fauna in Scottish waters, and is often caught as bycatch from the common lobster fishery grounds. This study aims to employ the species in relation to the biomonitoring potential for Mn in the Scottish inshore waters and in UK monitoring programmes by choosing Loch Fyne in the west coast of Scotland as a naturally high Mn area and the Clyde Sea area as the reference area. The ability of the crushed carapace to remove Mn from aqueous solution in a biosorption column system in the remediation of contaminated waters was also investigated as an attempt to turn this un-commercial species into beneficial use. Measured using standard atomic absorption spectroscopy (AAS), the concentrations of Mn in the tissues of the swimming crab L. depurator from Loch Fyne were consistently higher than in the tissues in crabs from the Clyde Sea area. The metal concentration differed according to sexes, and to the tissues in the order of the exoskeleton (carapace, gills) > hepatopancreas > crusher claw muscle and gonads. The trend observed in L. depurator was comparable to the shore crab, Carcinus maenas which is an established biomonitor for metals collected within the vicinity of both study areas. A series of different exposures of L. depurator to Mn in sea water (10ppm and 20ppm) for up to 21d, followed by a depuration period of 47d were performed under controlled laboratory conditions. Temporal changes in Mn concentrations in the exoskeleton of individual crabs were monitored by autotomizing a walking leg at weekly intervals. Mn concentrations in other tissues at given sampling points were obtained by sacrificing a batch of crabs at each sampling time. The hard tissues (dorsal carapace and leg exoskeleton) irreversibly accumulated Mn from the water whereas the soft tissues both accumulated Mn when exposed, and eliminated Mn after a period of depuration in clean sea water. As a result, the use of the exoskeleton of autotomized legs to represent Mn accumulation in the whole exoskeleton of a crab was established, and the accumulation of Mn from the water into the crabs tissues particularly the exoskeleton was confirmed. The ability of dried and crushed carapace particles from the swimming crab L. depurator to remove Mn from aqueous solutions was studied using a packed bed up-flow biosorption column system. From a batch experiment carried out at room temperature on fine carapace particles with a diameter of less than 300µm and 100ml of 80ppm Mn in distilled water, the data fitted the Freundlich adsorption isotherm with an adsorption capacity KF=22.82 mg.g-1. The breakthrough curves generated from a series of up-flow biosorption experiments (constant flow rate of 100ml.h-1, 72h sorption) indicates the great potential of the crab carapace particles to remove Mn from a solution. The removal depended greatly on the initial concentrations of the solution and the amount of carapace particles used which can be expressed as mass or the height of the column beds. Greater column bed heights increase Mn removal capacity which in the columns could be observed visually through progressive colour change of the beds. Experiments repeated using the carapace particles of the Norway lobster, Nephrops norvegicus (L.) generated similar trends and comparable data with the ones observed for L. depurator. Scanning electron microscopy (SEM) and SEM combined with electron dispersive analysis of x-ray (SEM-EDX) were applied to examine the morphology of the dorsal carapace and gills of L. depurator, and also the form and site of Mn deposition onto both the intact carapace of L. depurator and onto Mn-biosorbed carapace particles. The dorsal carapace of L. depurator resembles the typical crustacean cuticle with three main layers, the epi-, exo- and endocuticles made up primarily by CaCO3. Mn deposited onto the carapace especially on exposed inner layers and broken edges of the carapace particles in the form of Mn-rich nodules, which in 72h could create a layer visually observed as blackening of the particles. Deposition onto the gill surface took the form of fine particles scattered on the lamellae. Exposure of an isolated carapace to 80ppm Mn solution indicated the barrier-effect played by the epicuticle to Mn deposition onto the external surface. This barrier was lost when the surface is abraded. The membranous layer on the internal side did not act as a barrier, and penetration of Mn up to approximately 50µm into the endocuticular layers was detected. These results correlated with AAS measurements which indicated that internal exposure of the carapace resulted in a three times higher concentrations of Mn compared with external exposure. Based on the wide distribution of L. depurator around the UK coast and its habit of resting on the bottom sediment, the results of this study propose L. depurator as a biomonitor species for Mn in the bottom water, particularly in the Scottish waters. Given the abundance of the species in common lobster fishery grounds in Scotland and often caught as bycatch in the trawls, L. depurator could provide a continued source of materials if the carapace is to be converted into a good Mn-removing agent in Mn contaminated waters.

No description available.

The timely and efficient movement of chromosomes to the spindle equator during mitosis is a prerequisite for accurate chromosome segregation. Recent work has shown that the majority of chromosomes are able to congress and biorientate almost instantaneously after nuclear envelope breakdown due to their position relative to the forming spindle. However, other mechanisms are required to facilitate the congression of chromosomes, which do not congress in this initial wave. Congression of these remaining chromosomes is mediated by multiple mechanisms including: (1) Kinetochore sliding along the microtubule lattice using the Kinesin-7 CENP-E, and (2) kinetochores biorientating near the pole and congressing through microtubule depolymerisation-coupled movement. Here, we show that the constitutive centromere associated network (CCAN) subunit CENP-Q is required for both mechanisms. CENP-Q is required to recruit CENP-E to kinetochores thus explaining the absence of lateral sliding in CENP-Q depleted cells. Because depletion or inhibition of the CENP-E motor does not affect depolymerisation-coupled pulling, we identify a CENP-E recruitment-independent role for CENP-Q in chromosome congression. Following congression we find that biorientated kinetochore movements require both CENP-Q and CENP-E dependent mechanisms. This suggests that as biorientated kinetochores congress they switch into a mode that requires CENP-E motor activity.

The ER is a network of membrane sheets and tubules connected via three-way junctions. Reticulons are integral membrane proteins responsible for shaping the tubular ER. There are four reticulons isoforms in animals, two in yeast and 21 isoforms in Arabidopsis. This study developed an expression profile for isoforms AtRTNLB1-18 by expressing these reticulons driven by their native promoters. The reticulons analysed localised to the ER and coexist in many tissues. Given this result it is possible that the large size of the Arabidopsis gene family may have evolved to afford functional redundancy. It is known that a knock-out of seed specific RTNLB13 does not disrupt the seed ER morphology. Is this due to functional redundancy? In this study knock-outs were made of two other reticulons found non-exclusively in the seed, RTNLB1 and RTNLB2. In accordance with the redundancy theory, there was no apparent disruption in the mutants ER morphology. Knock-out/knock-down mutants of RTNLB1, RTNLB2 and RTNLB13 were also made and verified. The results of this were beyond the time frame of this study. In mammals and yeast reticulons are known to interact with other tubule forming proteins (DP1/Yop1p) and human atlastin. The Arabidopsis homologue of Yop1p is HVA22. In this study the seed-specific isoform HVA22b labels the tobacco ER (and the nuclear envelope), but it does not alter ER morphology or cause constrictions of the tubules (as seen with RTNLB13), suggesting that it is not a structural component. The closest plant homologue of atlastin is RHD3. This study shows that RL2, an RHD3 isoform that is highly expressed in the seed, locates to the ER without perturbing the ER morphology or Golgi body mobility. Over-expression of RL2 bearing mutations within its GTPase domain, however, induces cable-like ER, suggesting that a functional GTPase domain is required for the formation of three-way junctions. Co-expression of RTNLB13 with RL2 resulted in a striking modification of the ER network. This alteration was independent of an active RL2 GTPase domain but required a functional reticulon. RL2 and its GTPase mutants co-immunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 interact and operate synergistically in modulating ER morphology.

Ecotoxicology is currently undergoing a revolution as the result of new technological advances in molecular biology, capable of finely resolving metabolic mechanisms associated with exposure. These high-throughput analyses can detail the evolutionary and ecological implications of exposure in non-model organisms, such as the earthworm, Lumbricus rubellus. This terrestrial sentinel has been observed across former mine sites that are highly contaminated with arsenic and have been found to mitigate toxicity at soil concentrations that cause mortality in unadapted individuals. This is indicative of the adaptive capacity of natural populations recently exposed to persistent and strong selection pressure. However, mechanisms surrounding adaptation to arsenic in L. rubellus have yet to be characterised, and so the effects of exposure are broadly reported with the aim of distinguishing resistance from phenotypic plasticity in natural populations. Unadapted earthworms were initially used to derive basal phenotypic variation associated with arsenic exposure. Variation in life-history parameters was observed among adult and juvenile L. rubellus, establishing relative sensitivity and population-level inferences. A systems biology approach was employed to describe molecular mechanisms associated with arsenic metabolism, encompassing transcriptomic and metabolomic analyses, underpinned by arsenic speciation. Insight into the genetic bases of arsenic resistance, which enable persistence of L. rubellus at highly contaminated sites, was sought. Recombinant inbred lineages derived from adapted populations, were cultivated and their phenotypes relative to arsenic exposure determined. Phylogeographic analyses were used to interrogate genetic variation among populations inhabiting former mine sites as well as proximal control sites. A mitochondrial marker defined cryptic species across the UK, but did not establish soil chemical profiles relative to clade occurrence. RADseq better resolved genetic variation at these sites, determining that soil geochemistry is strongly associated with genetic variation. Furthermore, genomic markers inferred genetic erosion, found to selectively reduce variation at sites relative to a single clade.

This study examined the ecological role of herbivory on coral reefs of the Saudi Arabian Gulf coast. Herbivory is a ubiquitous process and important in regulating benthic marine communities. Three reef sites were studied over a 12-month period; one on an inshore fringing reef (1.5 m depth), and two on the fringing reef surrounding an offshore island (3 m and 11 m depth). The inshore site experienced greatest extremes in temperature, salinity and sedimentation. The ecological role of herbivory was determined from algal settlement plates and their selective exclusion from herbivores. The inshore site was naturally dominated by filamentous algae, while both offshore sites supported a higher proportion of crustose forms. Location (i. e. distance from shore) appeared to be more important than seasonality in determining the structure and composition of the epilithic algal community. At the inshore site herbivorous fish (dominated by Siganus spp. ) imposed a uniform, wide-ranging grazing pressure of intermediate intensity. Herbivorous echinoids (Echinometra mathaei) imposed an intensive but localised grazing pressure. At the shallow offshore site, only herbivorous fish (dominated by Scarus sp. ) appeared responsible for grazing impacts, which were also intensive. At the deep offshore site both herbivorous fish (dominated by Pomacentrus spp. ) and echinoids (Diademo setosum) were responsible for limiting algal growth, although other factors (e. g. light penetration) may also inhibit algal productivity at this site. An experiment assessing the effects of extreme perturbations (i. e. removal of the algal community), showed that seasonal life-history strongly affected generic succession and rate of re-colonisation. Perturbation effects were temporary and did not precipitate permanent alternative stable communities. An important secondary effect of herbivory is bioerosion. The mean erosion rate by E. mathaei was comparable to rates recorded elsewhere. Behavioural studies revealed that burrow defence and fidelity were positively correlated with burrow complexity, and that the frequency of agonistic behaviour was low. Foraging range was negatively correlated with burrow complexity. In addition, the risk of mortality by finfish predators at the inshore site was estimated to be very low. The study has shown that Saudi Arabian Gulf reef communities may be particularly vulnerable to the depletion of herbivores, for example by overfishing. Management plans should therefore safeguard the herbivorous community, in order to maintain natural bioerosion rates and other reef processes.