Previous studies have shown that poor maternal nutrition during pregnancy may induce metabolic syndrome in adults. In this study, we explored the mechanisms of how maternal low protein diet during the first 3.5 days of pregnancy, programs embryo development and embryonic stem cell function. Our results showed that mouse maternal low protein diet (9% casein; Emb-LPD) enhanced both Clathrin dependent and independent endocytosis in both in vivo blastocysts and in vitro differentiated embryoid bodies (EB day 5) compared with maternal normal protein diet (18% casein; NPD) controls. This increase in endocytosis was accompanied by an increase in lysosome volume per cell. This was done by confocal microscopy and 3D image analysis. To determine whether this effect on the lysosome system was due to autophagy or simply due to increased endocytosis, we studied the expression of LC3 protein, Clathrin, Megalin (also named as low density lipoprotein receptor 2 (LRP2)) and Cubilin. Immunostaining and Western blot analysis revealed Megalin and Cubilin were significantly up-regulated in Emb-LPD embryos and EBs, whilst Clathrin protein level was marginally increased and LC3 protein unaltered. This enhanced nutrient uptake ability was maintained even after cells or embryos were re-introduced into a normal environment in vitro. Thus, stimulated nutrient uptake in day 5.5 EB showed compensatory growth, known to associate with long-term disease symptoms. To understand the mechanisms involved, we investigated elements of the mTOR pathway. In vitro culture of early embryos in the presence of reduced levels of the three branched-chain amino acids (Lecine, Valine and Isoleucine) as occurring in Emb-LPD uterine fluid resulted in stimulated endocytosis of Trophectoderm (TE). In addition, we found although mTORC1 was partially suppressed, mTORC2 downstream RhoA-Actin interaction was stimulated in blastocysts by observing more actin and RhoA protein in Emb-LPD blastocysts as well as that inhibiting RhoA function abolished the enhanced endocytosis by Emb-LPD. We also investigated epigenetic changes induced by Histone deacetylase 3 (Hdac3) in terms of regulation of genes involved in Extraembronic Endoderm (XEN) differentiation and cardiomyocyte differentiation. We found that Emb-LPD EBs expressed reduced Gata6 and exhibited increased histone deacetylation at promoter of Gata6, together with increased Hdac3 expression. Our results reveal for the first time at the cellular level how early embryos respond to poor nutrition environment and reprogram to protect fetal growth. This further helps us to understand the mechanism of how adult metabolic syndrome can be originated from environment which early embryos were exposed to.

In Systems Biology, it is usual to use a set of ordinary differential equations to characterize biological function at a system level. The parameters in these equations generally reflect the reaction or decay rates of a molecular species, while states characterize the concentration values of species of interest, e.g. mRNA, proteins and metabolites. Often parameter values are estimated from in vitro experiments which may not be true reflections of the in vivo environments. With internal states, some may not be accessible for experimental measurement. Hence there is interest in estimating parameter values and states from noisy or incomplete observations taken at inputs/outputs of a system. This thesis explores several probabilistic inference approaches to do this. The study starts from a thorough investigation of the effectivenesses of the most commonly used one-pass inference methods, from which the non-parametric particle filtering approach is shown to be the most powerful method in the sequential category. After this study, the family of Approximate Bayesian Computation (ABC) methods, also known as likelihood-free batch approach, is reviewed chronologically and its advantages and deficiencies are summarized via a statistical toy example and two biological models. Additionally, a novel ABC method coupled with the sensitivity analysis technique has been developed and demonstrated on three periodic and one transient biological models. This approach has the potential to solve problem in high dimension by selectively allocating computational budget. In order to assess the capability of the proposed method in real-world problems, we have modeled the polymer pathway and conducted quantitative analysis via the proposed inference approach.

Voltage gated potassium channels (Kv channels) are tetrameric ion channels, responsible for regulating the potassium component of the membrane potential in a large range of cell types ranging from mammalian excitable cells to bacteria. Attempts have been made to elucidate the structure of voltage gated potassium channels using X-ray crystallography, however due to the inherent flexibility of the voltage sensors, removal of the channels from their native lipid environment causes distortion of the channels, and as a result much controversy remains over their exact structure. KvAP is a voltage gated potassium channel from the thermophilic archaea Aeropyrum pernix which contains a single cysteine residue, which can be removed by site directed mutagenesis to give a template for cysteine scanning mutagenesis. Fluorescence spectroscopy utilising cysteine reactive probes can be used to probe the membrane topology of proteins in the context of a lipid bilayer. Single cysteine mutants within the pore domain outer helix (S5 helix) of KvAP were generated and labelled with thiol reactive fluorescent probes. These probes were used to report on the polarity of the surrounding environment using a combination of the environmental sensitivity of the probes and fluorescence quenching from both aqueous and lipid phases. Fluorescence results fit well to a hypothetical model describing a trough like variation in dielectric constant of the membrane, allowing the determination of the position of the hydrophobic interface of the membrane at each end of the helix. A mutant of KvAP with no voltage sensing domains was also generated and subjected to cysteine scanning mutagenesis of the S5 helix. Again results fitted well to a hypothetical profile of the dielectric constant of the membrane, and the shift in fluorescence properties at some positions within the helix in the absence of the voltage sensor shows the residues of the pore domain which are in close contact with the voltage sensor.

A very important objective of ecological research is to explain the evolution of life histories, more specifically how natural selection modifies reproduction and development in order to generate the patterns that are observed in nature. With few exceptions, the reproductive mechanisms and patterns found in deep-water echinoderms are entirely similar to those found in shallow-water species. The aims of this study were 1) to examine the reproductive biology of the many deep-sea asteroids found on the continental slope to the west of Europe in order to determine if the reproductive adaptations are a function of depth, distribution or are phylogenetically controlled, and 2) to conduct experiments on the effects of pressure and temperature on larval development of Atlantic asteroids, to investigate the physiological potential for deep sea invasion by shallow-water species. Eggs of the shallow-water asteroids Asterias rubens Linnaeus and Marthasterias glacialis (Linnaeus) were fertilized in vitro and incubated through the early embryonic cleavages until the larval stage. They were subjected to different temperature/pressure regimes. Early embryos were able to tolerate pressures up to 150 atm at 15oC and 100 atm at 10oC. Survivorship of A. rubens swimming bipinnaria remained high (> 70%) after incubation at all the pressure/temperature combinations. In M. glacialis the highest survival of swimming larvae was 100% at 1 atm/5, 15 and 20oC and 50 atm/15 and 20oC. Data for the temperature and pressure effects on the later stages of development suggest that all the larval stages are more temperature/pressure tolerant than the early embryos and survivorship becomes greater with larval age. Therefore, the larvae of these two species could survive transport to deeper waters and these species may be capable of sending colonists to the deep sea. In the deep NE Atlantic the habitat has selected for species with specific reproductive traits, which provide them with successful and advantageous life history strategies. This can be clearly observed in the upper bathyal zone between 700 and 1100 m, where the environmental conditions have selected for small species with low fecundity and large eggs, plus habits related directly or indirectly with suspension feeding. These species exhibit reproductive features with trends to the opportunistic strategy and are distinctive of unpredictable environments, although their large egg size probably follows the general trend observed in species from cold waters in order to provide the larvae with energy sufficient for a high survival possibility. Conversely, phylogenetic and evolutionary factors are also important and seem to be decisive at the deepest waters where basically mainly species belonging to the strict deep-sea family Porcellanasteridae are found. All these species possess a mixture of features typical of classical K strategists and equilibrium strategists, which enable them to persist in a relatively stable environment with low energy availability. A comprehensive knowledge of the reproductive processes of the deep-sea fauna is essential in order to evaluate the level of variability caused in the environment principally by human activity and the possible effects on life-history of the species.

Abundance-size distributions of organisms within a community reflect fundamental properties underlying population dynamics. These include characteristics such as predator to prey biomass ratios, given the relationships that exist between body mass and metabolic activity, and between body mass and the ecological regulation of population density. In this way, plankton size has an important role in structuring the rates and pathways of material transfer in the marine pelagic food web, and consequently the oceanic carbon cycle. The transfer of energy between trophic levels can be inferred from regular patterns in population size structure, where plots of abundance within size classes, also known as plankton size spectra, typically show a power-law dependence on size. Metabolic theory, based on such size relations, has provided the basis for using an allometric approach to investigate the metabolic balance of the Atlantic Ocean and to identify the main drivers of trophic status in the plankton community. Samples were collected during three Atlantic Meridional Transect (AMT) cruises with further samples from a Marine Productivity (MarProd) cruise in the Irminger Sea. Three image analysis instruments were used to obtain plankton size spectra in the pico- to mesozooplankton size range. Data from a decadal time series at a coastal station off Plymouth, UK additionally enabled seasonal trends in plankton size spectra to be interpreted. Allometric relationships were also derived from physiological rates of individual plankton and scaled from organisms to ecosystems using community size structure data that were obtained from six earlier AMT cruises. Contrary to common perception, the transfer efficiency between phytoplankton and mesozooplankton in the Atlantic was not related to ecosystem productivity in oceanic and coastal systems. The flow of carbon up the food web was controlled by how quickly the consumers are able to respond to a resource pulse. These findings have fundamental implications for upper ocean carbon flux and suggest that global carbon flux models should reconsider the differences in carbon transfer efficiency between productive and oligotrophic areas of the world’s ocean. The allometric models of microbial community respiration and production provide a complementary method for understanding the metabolic balance of the upper ocean. Respiration exceeded photosynthesis in large areas of the Atlantic Ocean, suggesting that planktonic communities act as potential net sources of CO2. Large-sized phytoplankton are suggested as the main drivers of the balance between net autotrophy and heterotrophy.

No description available.

Dispersal and connectivity of patellid limpets (Patella spp.) in the eastern North Atlantic have been examined by addressing reproductive biology, larval development, population genetics and physical modelling of dispersal. The reproductive cycles of four limpet species were assessed on the northern and central Portugese coast, to determine spawning periods. This information was incorporated into dispersal models. The results showed that P. depressa and P. ulyssiponensis have almost year-round breeding, with a brief resting phase in the early summer. Conversely, the two other species displayed much shorter spawning periods, with gamete release taking place between December and March in P. vulgata and between September and December in P. rustica. The relationship between temperature and planktonic periods in P. depressa, P. ulyssiponensis, and P. vulgata was investigated with laboratory rearing experiments. Average duration of precompetent periods varied inversely with temperature, ranging between 3.7-14.0 days in P. depressa, 2.8-13.7 days in P. ulyssiponensis and 5.7-14.6 days in P. vulgata, whilst delay periods ranged between 15.8-25.4 days in P. depressa, 14.5-27 days in P. ulyssiponensis and 16.5-25 days in P. vulgata. Population genetic structure was examined on a range-wide scale in P. depressa and along the Iberian coast in P. rustica using microsatellite markers, plus one mtDNA locus in P. rustica. Results suggested high levels of gene flow throughout the study area and widespread lack of population differentiation in both species. A biophysical model of dispersal has been developed to assess the degree of demographic connectivity over ecological and evolutionary time frames, and to identify possible barriers to dispersal for P. depressa and P. rustica. The model predicted high levels of connectivity through most of the study area in both species, but in P. depressa simulations identified two large extensions of adult habitat discontinuity as barriers to larval dispersal. The model also showed that despite the potential for long-distance dispersal, most of the larvae released at one given location settle within much shorter distances. These results illustrate the need to view the study of marine dispersal as a multidisciplinary task, and suggest that relying on just one line of evidence may produce misleading results.

Neuropeptide Y (NPY) is widely expressed in both the central and peripheral nervous system and has an important role in the regulation of adult hippocampal neurogenesis by mediating the proliferation of neural precursor cells in both health and disease. The mechanisms underlying this neuroproliferative effect of NPY, however, are unknown. The aim of this project was to investigate these cellular pathways and the possible involvement of nitric oxide (NO) in NPY-mediated neuroproliferation using postnatal rat hippocampal cultures in vitro. NPY was found to have a purely proliferative effect on hippocampal neural precursor cells. The role of NO was explored by inhibiting the NO synthesising enzyme, nitric oxide synthase (NOS), which abolished the proliferative effect of NPY and supported the involvement of NO in NPY-mediated proliferation. Pharmacological analyses using subtype-selective inhibitors suggested that the neuronal isoform of NOS is the sole NOS subtype involved, which was expressed by both nestin+ precursors and class III β-tubulin+ neurons, the cell types previously shown to be responsive to NPY. The involvement of NO was further verified through loading hippocampal cells with an NO indicator, diaminofluorescein diacetate, where an increase in NO/N2O3 production was observed in nestin+ precursors and class III β-tubulin+ neurons in response to NPY treatment. The downstream signalling pathways coupling NPY-mediated NO synthesis to cell proliferation were identified, through the use of selective pharmacological agonists and antagonists, as soluble guanylate cyclase, cGMP-dependent protein kinase (PKG) and the extracellular signal-regulated kinases (ERK) 1/2. By assessing levels of NPY-mediated ERK 1/2 phosphorylation in response to NOS inhibition, it was found that ERK 1/2 activation was mediated only via NOS/NO mechanisms. This proliferative cGMP-PKG-ERK 1/2 signalling cascade appears to be mediated by intracellularly released NO, while on the other hand, the addition of extracellular NO through the application of NO donors exerted an inhibitory effect on neural precursor cell proliferation. In addition to demonstrating the dual nature of NO, this is the first time that the signalling mechanisms underlying the proliferative effect of NPY on neural precursor cells have been described. Understanding the mechanisms underlying the proliferation of neural precursor cells will ultimately be beneficial by allowing the development of novel therapeutic interventions for promoting hippocampal neurogenesis. To analyse the role of NO in the NPY-mediated neuroproliferation of hippocampal cells in three-dimensional (3D) cultures, Laponite, a novel synthetic silica hydrogel, was used. Culture medium-based Laponite hydrogels were developed before cell viability within the hydrogels were assessed by culturing hippocampal monolayers under gel cover. Hydrogel cover, however, resulted in cell behaviour reminiscent of preservation/fixation as monolayers showed no spatial or morphological changes over time, with one possible explanation being the high gel osmolarity. Although attempts at cell seeding showed more positive results, with cells adhering to a low heavy metal content variation of the hydrogel, determination of cell viability remained a problem due to prominent dye-gel binding. Although the rheological properties of Laponite make its use attractive, the biocompatibility of the hydrogels with hippocampal cells still require further optimisation if they are to be used as cell culture matrices.

Nanostructures engineered from DNA are becoming increasing popular in interdisciplinary research laboratories globally due to their ease to design and build, as well as their huge potential as devices for applications such as drug delivery and biosensors. Although many studies have used these structures alongside molecules that interact with DNA, little has been done to understand the effects of nanostructured DNA on ligand binding, and vice versa. In this thesis, a simple DNA tetrahedron, folded from four oligonucleotides, is used as a model in which to assess ligand interactions for a variety of DNA-binding agents. We characterised two DNA tetrahedron designs by band shifts, cleavage assays, chemical probing and melting studies. One of these tetrahedra, based on a previous design, was modified to incorporate single binding sites on each edge for a number of sequence-specific DNA-binding ligands: minor groove binders Hoechst 33258 and distamycin, intercalators actinomycin D, echinomycin, TANDEM and nogalamycin, and major groove binding triplex forming oligonucleotides. Band shifts, DNase I footprinting, global melting with UV and SYBR green I, as well as location-specific melting with fluorescent beacons were used to assess the interaction of these ligands with the DNA tetrahedron compared to linear duplex DNA. The results found actinomycin was able to inhibit folding of the structure, whereas other ligands did not. Sequence-selectivity was generally consistent across linear duplex and nanostructured DNA, though some changes in secondary binding sites were observed, however, Hoechst, distamycin and actinomycin showed reduced affinity to the nanostructure. Global thermal melting studies showed that all the ligands enhance stability, however, fluorescence beacons placed at specific locations found that many of the ligands were able to stabilise regions of the tetrahedron remote from their binding sites, including across junctions. The work described helps present a clearer understanding of ligand interactions with DNA nanostructures, as well demonstrates a model in which to assess structure stability at specific locations within a DNA nanostructure. This will aid the design and function of future DNA-based devices.

No description available.