Thesis (Ed.M.)--Boston University

Stem cells have potential use in tissue engineering and regenerative medicine, and as they underlie the development and maintenance of tissues throughout life, how they function is also of interest. The extracellular matrix presents a variety of physical and chemical signals to stem cells to regulate their behaviour in vivo. Recapitulation of these signals in vitro could enable the control of explanted stem cells to facilitate their study. Biomaterials that display extracellular-matrix inspired cues are one way to do this. By combining surface chemistry and fibronectin, an extracellular matrix protein with cell binding and growth factor binding domains, the conformation of fibronectin was controlled to create artificial extracellular matrices. Adsorbed on a film of poly(ethyl acrylate), fibronectin adopted a network-like conformation which ostensibly increased the exposure of its functional domains, whereas on poly(methyl acrylate) it had an unconnected organisation with more concealed domains. The growth factors bone morphogenetic protein 2 and vascular endothelial growth factor, known to bind to fibronectin, were adsorbed to the network conformation. Prior studies have reported that these artificial extracellular matrices differentially affected cell behaviour. In this work, the growth and differentiation of human bone marrow stromal cell surface marker-1 positive mesenchymal stem cells was characterised on these substrates. It was shown that all combinations of fibronectin conformation and growth factors supported cell adhesion and growth. A high-content image processing and analysis pipeline was developed to take advantage of automated fluorescence microscopy to show that cytoskeletal, nuclei, and differentiation-associated protein features distinguished cells cultured on the artificial extracellular matrices. Those on the isolated conformation and the network conformation with vascular endothelial growth factor were particularly distinct. Further, metabolomics revealed several metabolic pathways that differed in activity between the fibronectin conformations. To analyse the metabolomics data a Quick Results web application was built, which extended the existing Polyomics integrated Metabolomics Pipeline. The application improves the visualisation and interpretation of untargeted liquid chromatography—mass spectrometry metabolomics data. This work gives insights into how these artificial extracellular matrices can control stem cell behaviour, and developed and demonstrated several tools to improve the understanding of these biomaterials and the use of metabolomics data.

The sand lizard (Lacerta agilis) is a rare, elusive and cryptic reptile species of conservation importance in the UK. Knowledge of its ecology and behaviour has limited the development of a reliable and effective methodology for population monitoring; this threatens to compromise conservation effort. The behaviour of sand lizards varies seasonally, with sex and with environmental conditions, none of which are fully understood. This aim of this thesis is to further our ecological knowledge of the sand lizard, specifically by investigating factors which influence the detection probability of this species and through exploration of population monitoring and estimation methods. The detection rate of both male and female lizards in a captive population was found to be heavily dependent on their sex and reproductive stage. The detection probability of males was higher (39%) before mating than after mating (33%): with pre-mated males this was strongly associated with temperature and time of day; and with post-mated males it was linked to changeable conditions with solar radiation values between 200-700Wm-2. The detection probabilities of females were higher after mating (40%) than before (25%) with additional variations in detection rate during and after egg laying. Pre-mated females were most commonly observed basking in a preferred range of ultra-violet light and post-mated they favoured conditions where the ground surface temperature ranged between 17.5oC and 27.5oC. An intensive capture-mark-recapture study was performed on independent sand lizard populations and pattern matching software was used to assist with the identification of individual sand lizards from their dorsal patterning. The low recapture rate of sand lizards proved problematic and population estimates were generated using Program MARK following amalgamation of the data from each field season: but this nonetheless generated the first estimates of sand lizard populations in the UK, with a maximum mean density of 222Ha-2. The survivorship of males was consistently higher than females (0.67 vs. 0.26) and the detection rates and populations estimates differed between years. A high number of individuals were encountered only once, resulting in wide confidence limits of abundance estimates for this species. The range of microclimates available within a structurally diverse array of heathland vegetation were quantified and found to be more extensive than previously thought. The range of microhabitats within the preferred temperature range for sand lizards increased from Spring to Summer, so the need for a lizard to be in the open, and thus available for detection decreased. The obstacles encountered when attempting to monitor sand lizards populations arise from the unpredictable nature of the species. Future sand lizard surveys should be conducted at times when the detection probability maximised, as described in this thesis. A national monitoring scheme should steer away from absolute abundance estimates of populations and consider the use of occupancy estimation to monitor our remaining populations.

This thesis presents advances made to the scanning ion conductance microscope (SICM), a tool predominantly used to date for topographical imaging of biological samples. This technique is demonstrated to be a powerful tool for non-invasive surface charge mapping as well, through probing of the diffuse double layer formed at charged interfaces. Surface charge mapping with SICM is demonstrated for a range of samples, including biological systems, and it is shown that through the use of a novel feedback technique, also introduced herein, and newly implemented scanning regimes, that the surface charge information can be elucidated unambiguously, together with topography. Through adopting a characterisation protocol presented in this work, which helps provide a fuller understanding of the used nanopipette probe, the SICM response to charged interfaces and also in bulk solution can become quantitative, allowing for surface charge values for cell membranes and other substrates to be determined. This combination of: SICM experiments, complete probe characterisation and FEM simulations serves as a robust platform for investigating biological and other charged interfaces. The surface charge mapping protocols used allow for unseen surface charge heterogeneities, presented on cell membranes, to be identified and are amenable to future studies, performed in combination with other microscopy techniques, that could help correlate charged domains with physiological function. Finally, the nanopipette probe is also used as a reaction centre for driving the crystallisation of calcium carbonate, as an exemplar system. Through partitioning the constituent ions of calcium carbonate, with calcium present in a bath solution, and carbonate ions in a nanopipette, a bias can subsequently be applied to drive the ions together, leading to the formation of a crystalline entity, which blocks the nanopipette. Changes in the nanopipette conductance can then provide information about the growth process or subsequently the dissolution as the applied bias is reversed. FEM simulations can allow for an understanding of the underlying mixing problem and the technique is shown to be powerful for the screening of growth additives.

Changes in modem agriculture have been shown to have detrimental affects on those bird species whose populations mainly inhabit the wider countryside. Recently extensive changes to the farmed landscape have occurred through implementation of European agricultural policy much of which has been concerned with decreasing agricultural production. Lapwing (Vanellus vanellus), with almost the entire British population breeding on farmland, is a species that is thought to have been particularly affected by agricultural change. A study attempting to assess the impact that modern, intensive arable farming had on a breeding population of lapwing was undertaken from 1995 to 1997 in south-west Lancashire. The study site had an open character and was predominantly winter farmed (cultivation occurring September-February)although more than a third of the area was spring cultivated. Permanent pasture and rotational set-aside (RSA) accounted for about 2% and 5% of the cropped area respectively. Lapwings preferred to nest in spring farmed fields and RSA. The overall nest survival was 55.5% and did not differ significantly between years. Hatching success was highest in RSA (88%), lowest in spring farmed fields (48.4%) and intermediate in winter farmed fields (57.2%). Hatching success was significantly higher in spring farmed fields than in other habitats but did not differ significantly between winter and spring farmed fields. The hatching success of winter farmed fields was elevated by the success of nests of fields cultivated late in the winter. The main cause of nest losses was farming operations which caused 77.4% of all nests lost. Predation accounted for 14.4% of nest losses and desertion for 8.2%. There were annual differences in nest destruction in winter farmed crops caused by differences in the timing of agricultural operations. Nest destruction was highest in unsown fields but was compensated for by a high rate of replacement and high hatching success in replacement clutches. Two types of crop were spring farmed, combinable (mainly cereals) and vegetable (mainly potatoes) crops. Higher hatching 2 success in spring cereals than in vegetables was related to the differing intensities of management between the two crop categories. Lapwing preferred to nest in dense aggregations and far from ditches both of which reduced the risk of predation. Low densities of nest predators were maintained in the study area by predator control. Chicks from larger eggs survived better than chicks from smaller eggs. Chicks undertaking movements from natal to rearing fields suffered high levels of mortality. Chick mortality was caused by poor body condition, entrapment in field boundary ditches or predation or from the interaction of these factors. Brood movements were influenced by the distribution of crop types. Spring farmed fields and pasture were used for rearing broods. Predation was the main proximate cause of mortality for radio-marked chicks and accounted for 52% of all losses. Predation was a significant mortality factor until chicks were at least 20 days old, whereas poor body condition (31 % of all radio-marked chick losses) and ditch entrapment (17% of all losses) only killed very young chicks. Fledging success or hatching success is thought to be the main limit on productivity. Dietary studies revealed chicks ate mainly beetles but their diet varied depending upon rearing location. Surface living chick prey was abundant throughout the season in arable fields and late-hatched chicks suffered higher mortality than those hatched earlier mainly due to an increase in predation late in the season. Recommendations to maintain or improve the conditions for breeding lapwing within the study site are discussed. They include proposals which could operate under the agrienvironment regulations of the CAP, such as an increase in the area spring farmed with the cultivation of both cereal and vegetables and changes to the management of rotational set-aside to make such fields more suitable for brood rearing.

No description available.

No description available.

Circadian rhythms, i.e. rhythms exhibiting a cyclic behaviour with a period of approximately 24 hours, are present in the metabolism of most living organisms. The transcriptional processes, i.e. the processes associated with mRNA synthesis, critically contribute to their origination, and are responsible for most of the mechanisms which regulate gene expression levels in cells. Inhibition or activation of a putative transcriptionally regulated ‘child’ gene can be achieved via binding of proteins called transcription factors (TFs) to the gene promoter, a region of the DNA containing protein-specific binding sites. In this work, we investigate modelling and inference approaches for different scenarios of circadian transcriptional regulation. We focus on a system which comprises two transcription factors and a regulated child gene. We first perform parameter inference in the context of state-space models on simulated data from a mechanistic stochastic model describing this scenario. Additionally, we investigate the effect of data aggregation across different cells, and derive the smoothing equations for a destructive sampling scenario. In the second part of this work, we consider a situation in which an important regulator of a child gene has not been observed. We apply our model to mRNA expression levels of a subset of circadian genes of the Arabidopsis Thaliana model plant. Inference is in this case aimed at estimating both the model parameters and the unobserved transcription factor profile. We compare a posteriori the inferred transcription factor profiles with available time-series data for one important circadian regulator in the Arabidopsis Thaliana, namely late elongated hypocotyl (LHY), and identify similarities for a several genes known to belong to the central clock. Finally, we focus on a scenario of transcriptional regulation which includes an auto-regulatory negative feedback loop. This modelling framework is motivated by the availability of spatio-temporal imaging data of genes belonging to the mammalian central clock in mice suprachiasmatic nucleus (SCN), and in particular here we focus on Cry1. We introduce a distributed delay to account for nuclear export, translation, protein complex formation, and nuclear import, of the molecular species involved. To perform inference, we develop a novel filtering algorithm that can be applied to any system with distributed delays. We finally apply the methodology to Cry-luc spatio-temporal data, and find that parameter estimates are spatially distributed, with a marked difference between central and peripheral SCN regions.

The objective of the work described in this thesis was to develop novel experimental and analytical methods to study samples of anisotropically oriented fluorophores. The primary development was the fluorescence detection method for linear dichroism spectroscopy (FDLD). The experimental configuration of the measurement has the detector placed facing the propagation direction of the exciting incident light. Long-pass edge filters, selected so that their cut-off wavelength lies between the excitation and emission maxima of the sample’s fluorophore, were placed between the sample and detector to block transmitted incident light, whilst allowing Stoke’s shifted fluorescence emissions to pass freely. The experiment was designed to be conducted using a commercial circular dichroism (CD) spectropolarimeter, which has been adapted to measure linear dichroism (LD). A theoretical framework to calculate FDLD spectra using the output of such an instrument is presented, and used to generate the FDLD spectra of small molecules oriented on stretched oxidised polyethylene films and biological samples oriented in Couette flow. It was found that much of the information that can be obtained from LD may also be derived from FDLD spectra, however, FDLD possessed two clear advantages: i) fluorescence detection is highly sensitive, which significantly lowered the sample volume requirement of a measurement, and ii) fluorescence detection is more selective than absorption methods, as only chromophores that are also fluorophores are detected. A method for the cloning, expression and purification of the Escherichia coli actin homologue MreB is also presented. The composition of secondary structure elements within the protein obtained was analysed using CD and found to be in good agreement with literature values, taken from the crystal structure of Caulobacter crescentus MreB. Fluorescence spectra of the protein were recorded, which indicated that it may be possible to study its in vitro polymerisation kinetics using FDLD. Unfortunately, we were unable to obtain our Escherichia coli MreB in a sufficient yield to develop this assay. Finally, a novel method for detecting the wall shear stress (WSS) exerted on a specific point of a surface by a fluid flowing over it is reported. This information was derived from the response to a shear flow of a fluorescently labelled particle of M13 bacteriophage, which was tethered to the surface. The focus of this thesis was primarily on the analysis of the fluorescence signals. Using a custom made algorithm, microscopy images of the tethered phage particle were segmented to define the region of each image occupied by the particle, and to calculate its orientation and length at all time points. These data were used to calculate the WSS at the point of the particle’s attachment, and show that it varied when the surface was not uniform — highlighting a potential problem with commonly used methods for determining WSS that average over surface dynamics, such as particle image velocimetry and particle tracking velocimetry.

Female-female competition has been relatively overlooked in favour of male-male competition for mates, but it can also have important reproductive consequences. There are an increasing number of studies describing conditions where females compete to obtain breeding rank, gain access to or control resources or actively defend young. Communally breeding females are thought to be relatively egalitarian, sharing the cost of parental care with other females. Hence little attention has been paid to the potential for competition in such breeding systems, despite evidence of aggression and reproductive suppression between females. This thesis therefore explores the extent of competitive behaviours between female wild house mice (Mus musculus domesticus), a species with communal care of young, and investigates the physiological effects of competition and its consequences for breeding success and reproductive output. I examined the effects of age and other characteristics that may predict the degree of female competition. I identified that body mass, relative age of social partners, urinary testosterone concentration and reproductive experience were all useful predictors for the amount of competitive behaviour observed between female pairs. Following competitive female interaction I found that urinary testosterone and protein output increased, but there was no significant change in body mass and no significant effect on oestrus cycle length. Older females (> 12 months) with competitive experience had larger adrenal glands compared to females previously housed with their sisters, suggesting a possible stress response to competitive interaction. There was also evidence that competitively housed females had enlarged clitoral glands, which may play a role in signalling social status. As female house mice were found to compete and assume social ranks, I investigated the impact of female social status on male mate choice and mating behaviour. There was no evidence of significant male preference for more or less competitive females prior to or after competitive interaction in a choice test with restricted access to females or when presented with female odours. To investigate breeding behaviour I introduced female pairs to a male in semi-naturalistic enclosures, filming continuously over a four day period to examine mating attempts and female behaviour. Interestingly males mounted less competitive females either exclusively or preferentially during the test, with a small number of competitive females interrupting mating behaviour between their social partners and the male. Therefore males may prefer female partners that are less likely to act aggressively towards their advances. The effect of female competition on reproductive success was examined by comparing breeding success of subjects under solitary and communal breeding conditions. Despite the prediction that reproductive success increases for secondary litters in house mice, reproductive output was significantly reduced for more and less competitive females in the communal nest compared to previous output in a solitary nest. This finding illustrates the negative impact of competition on reproductive success. Females that gave birth first in communal nests also had significantly fewer pups present on post natal day one compared to females that gave birth second. Interestingly female offspring of more competitive females in this experiment went on to produce larger litters on average than females born to less competitive females. Litters were also likely to be male biased if females had been reared in a competitive environment rather than a solitary nest, suggesting that competitive ability and rearing environments can both influence reproductive success for offspring. These results, together with evidence in the literature, suggest that competition does occur between communally breeding females, and that reproductive success can be affected as a result. However competition between communal females may be less intense than between females in cooperative systems, where reproductive skew is biased towards one or two individual females in a group. Using a comparative analysis I found that cooperatively breeding species had increased reproductive output compared to other polytocous species, which is likely to be influenced by the presence of non-breeding helpers in the nest site. Cooperative species also had decreased inter-litter intervals compared to non-cooperative species, as well as a reduction in lactation length and protein content of milk. Communal species were found to have increased offspring growth and reduced sexual size dimorphism, suggesting that competition between females may have resulted in selection for increased female body mass. Together these results illustrate the significance of female competition in wild house mice, with consequences for mating behaviour and reproductive success, as well as the evolutionary implications of female-female competition in mammalian species residing in communal breeding systems.